Method of processing a biological and/or chemical sample

ABSTRACT

The invention provides a method of processing a biological and/or chemical sample. The method includes providing a fluid droplet, which includes an inner phase and an outer phase. The outer phase is immiscible with the inner phase, and the outer phase is surrounding the inner phase. The inner phase includes the biological and/or chemical sample. The fluid droplet furthermore comprises magnetically attractable matter. The method also includes providing at least one surface, which is of such a texture and such a wettability for the fluid of the inner phase of the fluid droplet, that the fluid droplet remains intact upon being contacted therewith. The method further includes disposing the fluid droplet onto the at least one surface. The method also includes performing a process on the biological and/or chemical sample in the fluid droplet.

FIELD OF THE INVENTION

The present invention relates to a method for processing a biologicaland/or chemical sample in a fluid droplet.

BACKGROUND OF THE INVENTION

Miniaturization of devices in the chemical, pharmaceutical andbiotechnological field has lead to the development of microfluidicdevices that control the flow of liquid and permit the performance of anumber of chemical and biological reactions. However, such devices donot allow downscaling a conventional, general-purpose chemistrylaboratory onto a single microchip due to the lack of appropriatemicrocomponents, such as microseparators or microfilters. Furthermore,such devices do often not meet mixing requirements. Therefore, anopen-well design, typically a multiwell-plate, is frequently employed incombination with automated mixing- and washing devices. However, such awell design poses increasing challenges upon further miniaturization andone of its major problems is evaporation.

The manipulation of droplets has recently received considerable interestdue to the possibility of isolating and handling volumes down to thepicoliter/femtoliter range (cf. e.g. international patent application WO2004/030820). Several lab-on-a-chip (LOC), micro total analysis (μTAS),and biological microelectromechanical systems (BioMEMS) have beendeveloped for moving, merging/mixing, splitting, and heating of dropletson surfaces, such as electrowetting-on-dielectric (EWOD) [Pollack, M. G.et al., Appl. Phys. Lett. (2000), 77, 1725-1726], surface acoustic waves(SAW) [Wixforth, A. et al., mstnews (2002), 5, 42-43], dielectrophoresis[Cascoyne, P. R. C. et al., Lab-on-a-Chip (2004), 4, 299-309], andlocally asymmetric environments [Daniel, S. et al., Langmuir (2005), 21,4240-4228]. However, these methods lack the most important operation forperforming sequential biological processes: the ability toseparate/purify/isolate starting material and/or reaction products fromcrude or complex mixtures. In order to permit such a separation a solidphase needs to be introduced as part of the droplet-based system.

Accordingly, it is an object of the present invention to provide amethod for processing a chemical and/or biological sample, which avoidsthese discussed disadvantages.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a method of processing abiological and/or chemical sample. The method includes providing a fluiddroplet. The fluid droplet includes an inner phase and an outer phase.The outer phase is immiscible with the inner phase. The outer phase issurrounding the inner phase. The inner phase includes the biologicaland/or chemical sample. The inner phase is shielded from the environmentby the outer phase. The fluid droplet furthermore includes magneticallyattractable matter. The method also includes providing at least onesurface. The surface is of such a texture, and such a wettability forthe fluid of the inner phase of the fluid droplet, that the fluiddroplet remains intact upon being contacted with the surface. The methodfurther includes disposing the fluid droplet onto the at least onesurface. The method also includes performing a process on the biologicaland/or chemical sample in the fluid droplet. In some embodiments themethod further includes controlling the position of the fluid dropletrelative to the at least one surface by exposing the fluid droplet to amagnetic or an electromagnetic field.

In a further aspect, the invention provides a fluid droplet. The fluiddroplet includes an inner phase and an outer phase, and at least onemagnetically attractable particle. The outer phase of the fluid dropletis immiscible with the inner phase of the fluid droplet. The outer phaseof the fluid droplet is surrounding the inner phase. The inner phase ofthe fluid droplet is shielded from the environment by the outer phase.The at least one magnetically attractable particle includes a ligandthat is capable of binding a biological and/or chemical sample.

In yet a further aspect, the invention provides a method of forming afluid droplet. The fluid droplet includes an inner phase, an outer phaseand at least one magnetically attractable particle. The method includesproviding a first fluid and providing a second fluid that is immisciblewith the first fluid. The method further includes contacting the firstfluid and the second fluid, thereby forming a fluid droplet thatincludes an inner phase and an outer phase. The first fluid is formingthe inner phase, surrounded by the second fluid forming the outer phase.The method further includes providing at least one magneticallyattractable particle. The magnetically attractable particle includes aligand that is capable of binding a biological and/or chemical sample.The method further includes disposing the at least one magneticallyattractable particle into the fluid droplet.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings, in which:

FIG. 1 depicts schematically various contact angles θ of a droplet on asurface (5), which is flat (A, B, C), convex (D) and concave (E). Thedroplet includes an inner phase (2) and an outer phase (3).

FIG. 2 depicts a droplet of a high contact angle θ on a surface (5) inside view (A) and top view (B). The droplet includes an inner phase (2)and an outer phase (3). The droplet also includes magneticallyattractable particles (1).

FIG. 3A shows a droplet of an inner phase (2) and an outer phase (3)that includes functionalized magnetically attractable particles (8) inside view. The position of the droplet below a surface (5) is controlledby means of a permanent magnet (20), in a position relative to thesurface (5).

FIG. 3B shows a droplet (6) of an inner phase (2), an outer phase (3)and magnetically attractable particles (8) on top of a surface (5).

FIG. 3C shows a droplet (6) as depicted in FIG. 3A or 3B on anelectromagnet (7), which is a member of an array of electromagnets seenfrom above or below respectively.

FIG. 4 depicts a washing process (cf. also FIG. 18) of a sample in adroplet (6) by means of a second droplet (16) in top view (A) as well asside view (B), depicting a magnet (20) under a surface (5). A seconddroplet involved in a washing process may be located at a surface thatdiffers from the surface (5), at which the fluid droplet that includesmagnetic particles (1), and an inner (2) and an outer phase (3) islocated (C).

FIG. 5 illustrates schematically the isolation of target matter from asample. A leukocyte (10) carrying a surface antigen (11) is bound by anantibody (9), coupled to a magnetically attractable particle (1).

FIG. 6 depicts a genetic analysis of a blood droplet sample using themethod of the invention. Leukocytes are bound to functionalizedmagnetically attractable particles (1) in droplets, isolated, washed,thermally lysed by means of thin film heaters (14) controlled by thinfilm sensors (15), and processed by reverse transcription (RT), followedby polymerase chain reaction (PCR) and pyrosequencing (PSQ). The arrowsindicate the direction, in which the sample is moved.

FIG. 7 depicts the isolation of white blood cells (WBCs), starting from0.1 μl fresh capillary whole human blood, which is mixed with a slurryof anti-CD15 and CD45-functionalized superparamagnetic particles (21),and incubated at room temperature for 10 min. After washing twice withPBS/1% BSA, the WBCs are ready for downstream applications.

FIG. 8 depicts DAPI-stained white blood cells (22) immobilized on top ofDynabeads CD15 and CD 45 after washing.

FIG. 9 depicts absolute numbers (left ordinate axis) and relative yield(right ordinate axis) of leukocytes isolated from a drop of human blood(▪), and 100 nl (); for 100 nl blood, the relative yield of isolatedleucocytes is 85% after 10 min.

FIG. 10 depicts the amplification analysis of a RT-PCR in a droplet byreal time detection.

FIG. 11 depicts a melting curve analysis of the obtained PCR products.One peak (T_(M)=87.6° C., value obtained using an Opticon 2 thermocyclerfrom MJ Research: T_(M)=84.6° C.) indicates one PCR product and littleco-products.

FIG. 12 depicts the verification of the identity of the cDNA fragmentamplified (FIG. 10) by capillary electrophoresis. 100 nl CD45/15 withabout 1400 white blood cells (▪) or a negative (no template) control(NTC. , grey), 1 μl PCR mixture and 5 μl mineral oil were used, thesample resulting in a PCR product of 208 bp (yield 20.5 ng/μl). 1:marker of 15 bp, 2: primer dimer, 3: marker of 600 bp.

FIG. 13 depicts a pyrogram analysis obtained by fluid-phasepyrosequencing of the PCR product (c_(dsDNA)=20.5 ng/μl).

FIG. 14 depicts a pyrogram analysis of the first three base positionsobtained by droplet-based pyrosequencing using the method of theinvention.

FIG. 15 depicts surface-bound fluorescein (FITC)-labelled goatanti-mouse IgG (whole molecule), covalently coupled on top ofMicromer®-M superparamagnetic particles (micromod).

FIG. 16 shows the colourless solution of a droplet (23) containing thesubstrate solution A (3,3′,5,5′-tetramethylbenzidine (TMB)) and B(hydrogen peroxide) turning dark (bluish/greenish, 24) after reactingwith the Gt×Ms IgG FITC/Rbt×Gt IgG Fc HRP-coated superparamagneticparticles. To stop the reaction, the superparamagnetic particles (25)are removed from the reaction mixture.

FIG. 17 depicts a temperature/time profile during PCR using the methodof the present invention. Due to the low thermal mass of the chip, whichis about 0.5 g, fast heating and cooling rates (±20-50 K s⁻¹) can becarried out.

FIG. 18 depicts further examples of performing a process on thebiological and/or chemical sample in a fluid droplet, which includemoving the fluid droplet, merging the fluid droplet with a further fluiddroplet, mixing the interior of the fluid droplet, washing the fluiddroplet by means of another fluid droplet, and splitting the fluiddroplet into daughter fluid droplets (cf. the Examples for details).

FIG. 19 shows an exemplary apparatus using an x, y-stage to manipulatethe fluid droplets in the method of the present invention. A Teflon AF(amorphous fluoropolymer)-coated glass slide, fixed by tape, is movedrelatively to a stationary permanent magnet (A), which is positioned onan x, y, z-stage (B, in detail).

FIG. 20 shows the content of the washing solution obtained by an initialwashing (A) and an additional washing solution obtained by a subsequentfurther washing (B) of the superparamagnetic particles-bound leucocytes.The additional washing solution hardly contains any erythrocytes. Itillustrates the efficiency of washing with a single droplet to removeerythrocytes, which is about five orders of magnitude.

FIG. 21 illustrates the mechanism of pyrosequencing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of processing a biologicaland/or chemical sample. The method is suitable for any process, inparticular a process that can be performed in a fluid on a miniaturizedscale (cf. below).

The sample may be of any origin. It may for instance, but not limitedto, be derived from humans, animals, plants, bacteria, viruses, spores,fungi, or protozoae, or from organic or inorganic materials of syntheticor biological origin. Accordingly, any of the following samples selectedfrom, but not limited to, the group consisting of a soil sample, an airsample, an environmental sample, a cell culture sample, a bone marrowsample, a rainfall sample, a fallout sample, a sewage sample, a groundwater sample, an abrasion sample, an archaeological sample, a foodsample, a blood sample, a serum sample, a plasma sample, an urinesample, a stool sample, a semen sample, a lymphatic fluid sample, acerebrospinal fluid sample, a nasopharyngeal wash sample, a sputumsample, a mouth swab sample, a throat swab sample, a nasal swab sample,a bronchoalveolar lavage sample, a bronchial secretion sample, a milksample, an amniotic fluid sample, a biopsy sample, a cancer sample, atumour sample, a tissue sample, a cell sample, a cell culture sample, acell lysate sample, a virus culture sample, a nail sample, a hairsample, a skin sample, a forensic sample, an infection sample, anosocomial infection sample, a production sample, a drug preparationsample, a biological molecule production sample, a protein preparationsample, a lipid preparation sample, a carbohydrate preparation sample, aspace sample, an extraterrestrial sample or any combination thereof maybe processed in the method. Where desired, a respective sample may havebeen preprocessed to any degree. As an illustrative example, a tissuesample may have been digested, homogenised or centrifuged prior to beingused with the device of the present invention. The sample mayfurthermore have been prepared in form of a fluid, such as a solution.Examples include, but are not limited to, a solution or a slurry of anucleotide, a polynucleotide, a nucleic acid, a peptide, a polypeptide,an amino acid, a protein, a synthetic polymer, a biochemicalcomposition, an organic chemical composition, an inorganic chemicalcomposition, a metal, a lipid, a carbohydrate, a combinatory chemistryproduct, a drug candidate molecule, a drug molecule, a drug metaboliteor of any combinations thereof. Further examples include, but are notlimited to, a suspension of a metal, a suspension of metal alloy, and asolution of a metal ion or any combination thereof, as well as asuspension of a cell, a virus, a microorganism, a pathogen, aradioactive compound or of any combinations thereof. It is understoodthat a sample may furthermore include any combination of theaforementioned examples.

Often, but not necessarily, the sample will include, or will be expectedto include, target matter or a precursor thereof. Such embodiments shallbe illustrated by a number of examples: The target matter may forinstance be a cell or a molecule added to or included in the sample, andit may be desired to obtain it in a purified or enriched form. Asanother example, the target matter may be a compound known or theorizedto be obtainable from a precursor compound by means of a chemicalprocess. In this case the sample may for instance include a solution ofsuch a precursor compound. As further example, a cell culture media maybe suspected to be contaminated. In this case, the method of the presentinvention may be used to identify the type of contaminant.

The target matter or precursor thereof may thus be of any nature.Examples include, but are not limited to, a nucleotide, anoligonucleotide, a polynucleotide, a nucleic acid, a peptide, apolypeptide, an amino acid, a protein, a synthetic polymer, abiochemical composition, a glycoprotein, a radioactive compound, apolyelectrolyte, a polycation, a polycatanion, a pathogen, an organicchemical composition, an inorganic chemical composition, a lipid, acarbohydrate, a combinatory chemistry product, a drug candidatemolecule, a drug molecule, a drug metabolite, a cell, a virus, amicroorganism or any combinations thereof. In embodiments where thetarget matter is for example a protein, a polypeptide, a peptide, anucleic acid, a polynucleotide or an oligonucleotide, it may contain anaffinity tag. Examples of affinity tags include, but are not limited tobiotin, dinitrophenol or digoxigenin. Where the target matter is aprotein, a polypeptide, or a peptide, further examples of an affinitytag include, but are not limited to, oligohistidine (such as a penta- orhexahistidine-tag), polyhistidine, a streptavidin binding tag such asthe STREP-TAGS® described in US patent application US 2003/0083474, U.S.Pat. No. 5,506,121 or 6,103,493, an immunoglobulin domain,maltose-binding protein, glutathione-S-transferase (GST), calmodulinbinding peptide (CBP), FLAG-peptide (e.g. of the sequenceAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Gly), the T7 epitope(Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly), maltose binding protein(MBP), the HSV epitope of the sequenceGln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virusglycoprotein D, the Vesicular Stomatitis Virus Glycoprotein (VSV-G)epitope of the sequence Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys, thehemagglutinin (HA) epitope of the sequenceTyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala and the “myc” epitope of thetranscription factor c-myc of the sequenceGlu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu. Where the target matter is anucleic acid, a polynucleotide or an oligonucleotide, an affinity tagmay furthermore be an oligonucleotide tag. Such an oligonucleotide tagmay for instance be used to hybridize to an immobilized oligonucleotidewith a complementary sequence. A respective affinity tag may be locatedwithin or attached to any part of the target matter. As an illustrativeexample, it may be operably fused to the amino terminus or to thecarboxy terminus of any of the aforementioned exemplary proteins.

In the method of the present invention the biological and/or chemicalsample is included in a fluid droplet, such as a liquid droplet. As anillustrative example, it may be included in an inner phase of such afluid droplet (cf. below). It may be deposited into the fluid droplet byany means (cf. below).

The method of the invention includes providing a fluid droplet. In afurther aspect, the present invention also relates to a fluid droplet asdescribed herein. As will become eminent in the following, a fluiddroplet as described herein functions as a self-organising virtualreaction chamber. The fluid droplet may be of any desired volume. It mayfor instance have a volume in the range of about 1 pl to about 1 ml, avolume in the range of about 0.1 nl to about 500 μl, or a volume in therange of about 100 nl to 100 μl. Handling of droplets of a volume above1 ml in air may in some embodiments require further adaptions of thedroplet environment. In this regards, the skilled artisan will be awarethat when using a droplet of large volume (such as e.g. 2 ml), therespective droplet may split into smaller droplets when contacting asurface. Where such splitting is undesired when carrying out the methodof the invention, suitable volumes for a droplet of a selected fluid caneasily be determined experimentally.

The fluid droplet includes magnetically attractable matter. Typicallyonly one phase of the fluid droplet contains magnetically attractablematter, i.e. either the outer or the inner phase of the fluid droplet.As an illustrative example, in some embodiments a magnetic fluid such asa ferrofluid may be included in the fluid droplet. A ferrofluid is forexample commercially available in form of a colloidal suspension ofsub-domain magnetically attractable particles in a liquid carrier fromFerrotec (Nashua, N.H., U.S.A.). A respective ferrofluid may forinstance be based on a non-polar liquid and form the outer phase of afluid droplet. In this case the inner phase may for instance be anaqueous solution. As a further illustrative example, an iron-richbacterium may be included in a phase of the fluid droplet. Manybacterial species contain iron as it is required for their metabolism. Alarge number, including Neisseria meningitidis and N. gonorrhoeae, havefor example transferrin and/or lactoferrin iron-uptake systems. Suchbacteria may only in certain embodiments contain sufficient iron to beused in the method of the present invention. Other bacteria reduce oroxidize iron and thus contain a higher amount thereof. An illustrativeexample of an (anaerobic) iron-reducing bacterium is Geobactermetallireducens. This bacterium may typically be included in an aqueousphase, whether the inner or the outer phase of a liquid droplet. Wherethe outer phase of the fluid droplet is for instance selected to be anon-polar liquid, this bacterium will typically be included in the innerphase of a respective fluid droplet (cf. also below). A respectivebacterium may for instance contain, e.g. by means of recombinantexpression techniques, a surface protein that is able to attract targetmatter.

As yet a further illustrative example, magnetically attractableparticles may be included in the fluid droplet. Such particles may beable to attract target matter. In some embodiments the magneticparticles can be functionalised with specific affinity for target matterand capturing target matter, therefore acting as a binding means (seebelow).

For convenience magnetically attractable particles are herein referredto as “magnetic particles” or “magnetic beads”. Magnetic particles maycontain diamagnetic, ferromagnetic, paramagnetic or superparamagneticmaterial. Superparamagnetic material responds to a magnetic field withan induced magnetic field without a resulting permanent magnetization.Magnetic particles based on iron oxide are for example commerciallyavailable as Dynabeads® from Dynal Biotech, as magnetic MicroBeads fromMiltenyi Biotec, as magnetic porous glass beads from CPG Inc., as wellas from various other sources, such as Roche Applied Science, BIOCLON,BioSource International Inc., micromod, AMBION, Merck, BangsLaboratories, Polysciences, or Novagen Inc., to name only a few.Magnetic nanoparticles based on superparamagnetic Co and FeCo, as wellas ferromagnetic Co nanocrystals have been described, for example byHütten, A. et al. (J. Biotech. (2004), 112, 47-63).

The magnetic beads may be designed to serve the function of attractingtarget matter through chemisorption, e.g. a covalent bond, orphysisorption, e.g. electrostatic attraction. The magnetic particlesused in such embodiments may provide a surface with an affinity forcertain matter allowing for instance to absorb/desorb proteins,peptides, nucleic acids and other compounds. Examples include, but arenot limited to, attractions by physical means, such as e.g. π-stacking,dipole-dipole, induced dipole-dipole, van-der-Waals, opposite charges,or H-bonding, e.g. antibody-antigen binding attractions, and affinityattractions formed between a ligand that has binding activity for thetarget matter and the target, such as for instance a ligand and a metal.As two further illustrative examples, physicochemical bonds, e.g.between gold and a thiol, or geometrical means, e.g. size exclusion, maybe relied on. Different areas of the same or several magnetic particlesmay also be designed to attract or “capture” the target matter.

In some embodiments the magnetic particles include a ligand that iscapable of binding target matter that is suspected or known to beincluded in the biological and/or chemical sample. Such a ligand may insome embodiments be capable of selectively binding such target mattersuch as, but is not limited to, an ion, a polyion, a metal, DNA, RNA, aprotein (including a synthetic analogue thereof), bacterial cells,spores, viruses, low molecular weight organic molecules, or inorganiccompounds. A respective ligand may be immobilized on the surface of theat least one magnetically attractable particle. FIG. 5 depictsschematically an example of target matter binding to a ligandimmobilized on a magnetic particle. The target matter is a leucocytecarrying a cell surface marker (e.g. CD45/15). The magnetic particleincludes γ-Fe₂O₃, Fe₃O₄ and polymethylmethacrylate-grafted polystyrol,immobilized thereon is an antibody directed against the cell surfacemarker (e.g. anti-CD45/15). FIG. 15 illustrates the binding of labelledtarget matter, a fluorescent antibody, to a magnetic particle.

A respective ligand may for instance be hydrocarbon-based (includingpolymeric) and include nitrogen-, phosphorus-, sulphur-, carben-,halogen- or pseudohalogen groups. It may be an alcohol, an organic acid,an inorganic acid, an amine, a phosphine, a thiol, a disulfide, analkane, an amino acid, a peptide, an oligopeptide, a polypeptide, aprotein, a nucleic acid, a lipid, a saccharide, an oligosaccharide, or apolysaccharide. As further examples, it may also be a cation, an anion,a polycation, a polyanion, a polycation, an electrolyte, apolyelectrolyte, a carbon nanotube, carbon nanofoam, a silica particle,a glass particle, or an alumosilicate. Generally, such a ligand has ahigher affinity to the target matter than to other matter. Examples of arespective ligand include, but are not limited to, a crown ether, anantibody, a fragment thereof and a proteinaceous binding molecule withantibody-like functions. Examples of (recombinant) antibody fragmentsare Fab fragments, Fv fragments, single-chain Fv fragments (scFv),diabodies or domain antibodies (Holt, L J et al., Trends Biotechnol.21(11), 2003, 484-490). An example of a proteinaceous binding moleculewith antibody-like functions is a mutein based on a polypeptide of thelipocalin family. See for example Beste et al., Proc. Natl. Acad. Sci.USA 96, 1999, 1898-1903 and WO 99/16873, WO 00/75308, WO 03/029471, WO03/029462, WO 03/029463, WO 2005/019254, WO 2005/019255 or WO2005/019256. Lipocalins described in these references such as the bilinbinding protein, the human neutrophil gelatinase-associated lipocalin,human Apolipoprotein D, human tear lipocalin, or glycodelin, possesnatural ligand-binding sites that can be modified so that they bind toselected small protein regions known as haptens. Other non-limitingexamples of further proteinaceous binding molecules are the so-calledglubodies (see WO 96/23879), proteins based on the ankyrin scaffold(Hryniewicz-Jankowska, A et al., Folia Histochem. Cytobiol. 40, 2002,239-249) or crystalline scaffold (WO 01/04144) the proteins described inSkerra, J. Mol. Recognit. 13, 2000, 167-187, and avimers. Avimerscontain so called A-domains that occur as strings of multiple domains inseveral cell surface receptors (Silverman, J, et al., (2005) NatureBiotechnology, 23, 1556-1561). Further examples of a suitable ligandinclude, but are not limited to, a molecular imprinted structure, anextracellular matrix, a lectin, protein A, protein G, a metal, a metalion, nitrilo triacetic acid derivates (NTA), RGD-motifs, dextranes,polyethyleneimine (PEI), polyelectrolytes, redoxpolymers, glycoproteins,aptamers, enzymes, a dye, streptavidin, amylose, maltose, cellulose,chitin, glutathione, calmodulin, gelatine, polymyxin, heparin, NAD,NADP, lysine, arginine, benzamidine, poly U, or oligo-dT. Lectins suchas Concavalin A are known to bind to polysaccharides and glycosylatedproteins. An illustrative example of a dye is a triazine dye such asCibacron blue F3G-A (CB) or Red HE-3B, which specifically bindNADH-dependent enzymes. Green A binds to CoA proteins, human serumalbumin, and dehydrogenases. The dyes 7-aminoactinomycin D and4′,6-diamidino-2-phenylindole bind to DNA. Cations of metals such as Ni,Cd, Zn, Co, or Cu, are typically used to bind affinity tags such as anoligohistidine containing sequence, including the hexahistidine or theHis-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys tag (MAT tag), thehexapeptide His-Ser-Gln-Lys-Val-Phe (binding cadmium), andN-methacryloyl-(L)-cysteine methyl ester. In addition a magneticparticle may be coated with a modifying agent that further increases theaffinity of the substrate for any or a certain form, class etc. oftarget matter.

In some embodiments the target matter is a molecule that is suspected orknown to be present within other (undesired) matter, from which it needsto be extracted. Extraction of a molecule from an organism, a part of anorganism, or an embryo may for instance include the usage of a compoundthat facilitates the transfer of a desired molecule from an organism ora part thereof into a fluid. An illustrative example of an extraction ofa molecule from a part of an organism is an extraction of proteins(wholly or partly) integrated into the cell membrane. It is oftendesired to transfer such proteins into an aqueous solution for furtherprocessing. A compound that facilitates the transfer of such proteinsinto an aqueous solution is a detergent. Contacting a respective cellmembrane with an aqueous solution, to which a detergent is added, willtypically result in an extraction of membrane proteins.

Where magnetic particles are used, they may at the same time as actingas a carrier for target matter, or alternatively thereto, themselves actas a tag or amplifier in the context of sensor technologies. Examplesinclude, but are not limited to, giant magnetoresistance (GMR) [Chiriac,H, et al., (2005) Journal of Magnetism and Magnetic Materials, 293,671-676], surface enhanced Raman spectroscopy (SERS), enhanced surfaceplasmon resonance (eSPR), and two-dimensional capillary electrophoresis.As an illustrative example, target matter may be bound to ligandsimmobilized on different magnetic particles in a fluid droplet accordingto the present invention. By means of further affinity ligands, whetherbound on a stationary phase, in solution, or otherwise the target mattermay be separated together with the magnetic particles bound thereto.Where the magnetic particles are exposed to a magnetic field, theydevelop a dipole field. This dipole field may be detected by a dipolesensor. By quantifying the amplitude of the sensor impedance the amountof target matter can be quantified.

The fluid droplet further includes an inner phase and an outer phase.The outer phase is surrounding the inner phase. In some embodiments theouter phase is a bulk phase accommodating the inner phase. In otherembodiments the outer phase is surrounding the inner phase as a film.The fluid of the outer phase may be a liquid or a gas. The fluid of theinner phase is typically a liquid.

In embodiments where the outer phase is a film, it is typically of avolume that is in the range of several magnitudes below to severalmagnitudes above the volume of the inner phase. The volume ratio of theinner to the outer phase may for example be selected in the range ofabout 1000:1 to about 1:1000, such as the range of about 10:1 to about1:10. As an example, for applications involving one or more liquiddroplets at room temperature it may be desired to chose a high volumeratio of the inner to the outer phase, for instance a ratio of about1000:1. For applications involving one or more liquid droplets in therange of about 100° C. it may be desired to choose a low volume ratio ofthe inner to the outer phase, for instance a ratio of about 1:1000. Insome embodiments a respective film is furthermore of uniform thickness.In other embodiments the film includes irregularities such as a cone.Respective irregular interfaces are for instance known for interfacesbetween water and some ionic liquids such as octylsubstitutedhexafluorophosphates.

The outer phase is immiscible with the inner phase. Typically, the fluidof the outer phase is immiscible with the fluid of the inner phase. Anyfluid may be used for the respective phase, as long as it is (a)immiscible with the other phase, so that two separate phases can form,and (b) the fluid does not prevent the desired process from beingcarried out. An illustrative example of two immiscible gases are heliumand carbon dioxide, which generally form two immiscible phases overextended composition ranges, as long as the temperature is below thecritical point of the binary mixture and the pressures is above thevapour pressure of pure liquid carbon dioxide. The process is typicallycarried out in the inner phase (cf. also below). Thus a selected fluidmay be of any property. In case a phase is selected to be a liquid or agas, it may for instance be a polar or a non-polar liquid or gas,respectively. Often liquids are classified into polar and non-polarliquids in order to characterize properties such as solubility andmiscibility with other liquids. Polar liquids typically containmolecules with an uneven distribution of electron density. The sameclassification may be applied to gases. The polarity of a molecule isreflected by its dielectric constant or its dipole moment. Polarmolecules are typically further classified into protic and non-protic(or aprotic) molecules. A fluid, e.g. a liquid, that contains to a largeextent polar protic molecules may therefore be termed a polar proticfluid. A fluid, e.g. a liquid, that contains to a large extent polarnon-protic molecules may be termed a polar non-protic fluid. Proticmolecules contain a hydrogen atom which may be an acidic hydrogen whenthe molecule is dissolved for instance in water or an alcohol. Aproticmolecules do not contain such hydrogen atoms.

Examples of non-polar liquids include, but are not limited to, hexane,heptane, cyclohexane, benzene, toluene, dichloromethane, carbontetrachloride, carbon disulfide, dioxane, diethyl ether, ordiisopropylether. Examples of dipolar aprotic liquids are methyl ethylketone, chloroform, tetrahydrofuran, ethylene glycol monobutyl ether,pyridine, methyl isobutyl ketone, acetone, cyclohexanone, ethyl acetate,isobutyl isobutyrate, ethylene glycol diacetate, dimethylformamide,acetonitrile, N,N-dimethyl acetamide, nitromethane, acetonitrile,N-methylpyrrolidone, methanol, ethanol, propanol, isopropanol, butanol,N,N-diisopropylethylamine, and dimethylsulfoxide. Examples of polarprotic liquids are water, methanol, isopropanol, tert.-butyl alcohol,formic acid, hydrochloric acid, sulfuric acid, acetic acid,trifluoroacetic acid, dimethylarsinic acid [(CH₃)₂AsO(OH)],acetonitrile, phenol or chlorophenol. Ionic liquids typically have anorganic cation and an anion that may be either organic or inorganic. Thepolarity of ionic liquids (cf. below for examples) is known to belargely determined by the associated anion. While e.g. halides,pseudohalides, BF₄ ⁻, methyl sulphate, NO₃ ⁻, or ClO₄ ⁻ are polarliquids, hexafluorophosphates, AsF₆ ⁻, bis(perfluoroalkyl)-imides, and[C₄F₆SO₃]⁻ are non-polar liquids. Each phase may also contain more thanone fluid. If for example more than one liquid is used for the inner orthe outer phase, the selected mixture of the liquids is still capable offorming a phase separate from the respective other phase of the droplet,and the liquids are generally miscible with each other in the selectedratio. As an illustrative example, ammonia, a polar gas, readilydissolves in water, a polar liquid, so that these two fluids may beincluded in a common phase.

Two immiscible phases may for instance be obtained where a polar fluid,such as a hydrophilic liquid (cf. below), is selected for one phase andnon-polar fluid, such as a hydrophobic liquid, is selected for the otherphase. As an illustrative example, carbon dioxide (CO₂), a non-polargas, does not dissolve (except for trace amounts) in water, a polarliquid. Under certain circumstances, such as under increased pressure,carbon dioxide may however be dissolved in water (cf. e.g. carbonateddrinks). In some embodiments the fluid of the inner phase may be a polarliquid and the fluid of the outer phase of the fluid droplet may be anon-polar liquid. Suitable polar liquids include, but are not limitedto, water, deuterium oxide, tritium oxide, an alcohol, an organic acid(including a salt thereof), an inorganic acid (including a saltthereof), an ester of an organic acid, an ester of an inorganic acid, anether, an amine (including a salt thereof), an amide, a nitrile, aketone, an ionic detergent, a non-ionic detergent, carbon dioxide,dimethyl sulfone, dimethyl sulfoxide, a thiol, a disulfide, and a polarionic liquid. Suitable non-polar liquids include, but are not limitedto, a mineral oil, a silicone oil, a natural oil, a perfluorinatedcarbon liquid, a partially halogenated, e.g. fluorinated, carbon liquid,an alkane, an alkene, an alkine, a cycloalkane, an aromatic compound,carbon disulfide and a non-polar ionic liquid.

As an illustrative example, the fluid of the inner phase of the fluiddroplet may be hydrophilic liquid and the fluid of the outer phase ofthe fluid droplet may be a hydrophobic liquid. Hydrophilic(“water-loving”) liquids, also termed lipophilic (“fat-loving”), containmolecules which can form dipole-dipole interactions with water moleculesand thus dissolve therein. Hydrophilic (“water-hating”) liquids, alsotermed lipophobic, have a tendency to separate from water. Examples of ahydrophilic liquid include, but are not limited to water, acetone,methanol, ethanol, propanol, isopropanol, butanol, tetrahydrofuran,pyridine, chloroform, ethylene glycol monobutyl ether, pyridine, ethylacetate, acetonitrile, dimethylformamide, N,N-dimethyl acetamide,N-methylpyrrolidone, formic acid, formamide, and a polar ionic liquid.Examples of a polar ionic liquid include, but are not limited to,1-ethyl-3-methylimidazolium tetrafluoroborate,N-butyl-4-methylpyridinium tetrafluoroborate,1,3-dialkylimidazolium-tetrafluoroborate,1,3-dialkylimidazolium-hexafluoroborate, 1-ethyl-3-methylimidazoliumbis(pentafluoroethyl)phosphinate, 1-butyl-3-methylimidazoliumtetrakis(3,5-bis(trifluoromethylphenyl)borate, tetrabutyl-ammoniumbis(trifluoromethyl)-imide, ethyl-3-methylimidazoliumtrifluoromethanesulfonate, 1-butyl-3-methylimidazolium methylsulfate,1-n-butyl-3-methylimidazolium ([bmim]) octylsulfate, and1-n-butyl-3-methyl-imidazolium tetrafluoroborate. Examples of anon-polar liquid include, but are not limited to mineral oil, hexane,heptane, cyclohexane, benzene, toluene, dichloromethane, chloroform,carbon tetrachloride, carbon disulfide, dioxane, diethyl ether,diisopropylether, methyl propyl ketone, methyl isoamyl ketone, methylisobutyl ketone, cyclohexanone, isobutyl isobutyrate, ethylene glycoldiacetate, and a non-polar ionic liquid. Examples of a non-polar ionicliquid include, but are not limited to, 1-ethyl-3-methylimidazoliumbis[(trifluoromethyl)-sulfonyl]amide bis(triflyl)amide,1-ethyl-3-methylimidazolium bis[(trifluoromethyl)-sulfonyl]amidetrifluoroacetate, 1-butyl-3-methylimidazolium hexafluorophosphate,1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide,1-butyl-3-methylimidazolium bis-(trifluoromethylsulfonyl)imide,trihexyl(tetradecyl)phosphonium bis[oxalato(2-)]borate, 1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate,1-butyl-3-methyl-imidazolium hexafluorophosphate,tris(pentafluoroethyl)trifluorophosphate,trihexyl-(tetradecyl)phosphonium,N″-ethyl-N,N,N′,N-tetramethylguanidinium, 1-butyl-1-methyl pyrrolidiniumtris(pentafluoroethyl) trifluorophosphate, 1-butyl-1-methylpyrrolidiniumbis(trifluoromethylsulfonyl) imide, 1-butyl-3-methyl imidazoliumhexafluorophosphate, 1-ethyl-3-methylimidazoliumbis(trifluoromethylsulfonyl)imide and 1-n-butyl-3-methyl-imidazolium.

A phase of the fluid droplet may include further matter, for exampledissolved, emulsified or suspended therein. As an illustrative example,where an aqueous phase is used, it may include one or more buffercompounds. Numerous buffer compounds are used in the art and may be usedto carry out the various processes described herein. Examples of buffersinclude, but are not limited to, solutions of salts of phosphate,carbonate, succinate, citrate, acetate, formate, barbiturate, oxalate,lactate, phthalate, maleate, cacodylate, borate,N-(2-acetamido)-2-amino-ethanesulfonate (also called (ACES),N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (also calledHEPES), 4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid (alsocalled HEPPS), piperazine-1,4-bis(2-ethanesulfonic acid) (also calledPIPES), (2-[Tris(hydroxymethyl)-methylamino]-1-ethansulfonic acid (alsocalled TES), 2-cyclohexylamino-ethansulfonic acid (also called CHES) andN-(2-acetamido)-iminodiacetate (also called ADA). Any counter ion may beused in these salts; ammonium, sodium, and potassium may serve asillustrative examples. Further examples of buffers include, but are notlimited to, triethanolamine, diethanolamine, ethylamine, triethylamine,glycine, glycylglycine, histidine, tris(hydroxymethyl)aminomethane (alsocalled TRIS), bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl)methane(also called BIS-TRIS), and N-[Tris(hydroxymethyl)-methyl]-glycine (alsocalled TRICINE), to name a few. The buffers may be aqueous solutions ofsuch buffer compounds or solutions in a suitable polar organic solvent.As an illustrative example, a buffer may be deposited in solid form, forexample freeze-dried. In such a case the solid buffer, e.g. a powder,may be dissolved in an aqueous phase by merging and or mixing, forinstance assisted or performed by means of ultrasound. In such a casethe amount of volume of a respective aqueous phase used may for instancebe used to obtain the desired final buffer concentration.

Further examples of matter included in a phase of the fluid dropletinclude, but are not limited to, reagents, catalysts and reactants, forcarrying out a chemical or biological process. As an illustrativeexample, salts, substrates or detergents may be added in order tomaintain cells or proteins in an intact state. As a further illustrativeexample, chelating compounds may be required, for instance to protectorganisms from traces of otherwise toxic salts or to increase the yieldof a chemical reaction. As yet further illustrative examples, protease,RNase, or DNase inhibitors may be added in order to maintain proteins,RNA, or DNA in an intact state. A further example of a possible additiveto a phase of the fluid droplet includes magnetically attractableparticles (see above).

The inner phase of the fluid droplet is shielded from the environment bythe outer phase. The outer phase may thus for example act as a barrieror as a seal. The term “environment” refers to any fluid or solidmatter, such as for instance a gas (of any desired density or pressure)or a liquid, which is not part of the inner phase, the outer phase or asurface, on which the fluid droplet is disposed. As an illustrativeexample, the outer phase may prevent or reduce evaporation of the innerphase into surrounding air. As a further example, the outer phase mayprovide a barrier in terms of contact or diffusion etc. The outer phasemay for instance prevent contact with solid matter such as sand or dustparticles or with fluid that would be miscible with the inner phase ofthe fluid droplet. The outer phase of the droplet may also provideaccess of energy, such as electromagnetic radiation of a certainwavelength to the inner phase. The outer phase may also serve inprotecting a surface at which the fluid droplet is positioned againstcontamination by components of the inner phase of the fluid droplet.Furthermore, the outer phase may enable a sample such as a body liquid,e.g. blood, sputum, etc. to move on a non-polar surface (e.g. PTFE). Insome embodiments the outer phase may also maintain sterility of theinner phase, even where the fluid droplet as a whole is being handledunder, or exposed to, non-sterile conditions. The outer phase mayfurthermore allow for the contact and fusion with another fluid dropletthat includes two phases of similar polarities (e.g. similarhydrophobicities). As an example, where the outer phase is a hydrophobicliquid and the inner phase is a hydrophilic liquid, the outer phase maybe capable of merging with the outer phase of a further fluid dropletthat is hydrophilic and surrounds an inner phase that is hydrophilic. Insuch a case a spontaneous fusion of the two exemplary droplets mayoccur.

The fluid droplet may be provided by any means. Forming the fluiddroplet includes providing the fluid of the inner phase, providing thefluid of the outer phase and providing the sample. The fluid dropletthat includes two phases, as used in the present invention, is aself-organizing system, the formation of which is driven by surfaceenergy. Accordingly the inner or the outer phase, or the sample may beprovided first, in a second or in a final step. Alternatively any one ormore (or parts) of the inner phase, the outer phase, or the sample maybe provided simultaneously. As an illustrative example, forming thefluid droplet may include providing a first fluid and providing a secondfluid that is immiscible with the first fluid. Forming the fluid dropletmay further include dispensing an aliquot, for instance a droplet, ofthe first fluid onto the second fluid, thereby forming one or more fluiddroplet(s) of the first fluid surrounded by the second fluid, therebyforming one or more fluid droplets comprising an inner phase and anouter phase. In some embodiments forming the fluid droplet may alsoinclude collecting the droplet of said first fluid, or a part thereof,out of said second fluid that is forming the outer phase. In this case adroplet may be formed that includes an outer phase surrounding the innerphase as a film. In some embodiments an initial larger droplet can beformed by forming a fluid droplet of the first fluid in the secondfluid. In these embodiments, from this initial larger droplet severalsmaller droplets can then be collected. Due to fluid droplet being aself-organizing system, there are generally no additional means requiredin order to achieve a respective portioning of an initial volume.

In this respect the present invention also relates to a method offorming a fluid droplet as defined above. The method includes forming afluid droplet as just described, i.e. by providing two fluids immisciblewith each other, contacting the first fluid and the second fluid,thereby forming a fluid droplet of a first fluid surrounded by a secondfluid (supra). In one embodiment of the method, contacting the first andthe second fluid includes dispensing a droplet of the first fluid intothe second fluid. In some embodiments the method may also includecollecting the droplet of the first fluid out of the second fluid,thereby forming a fluid droplet that includes an inner phase and anouter phase, the latter surrounding the inner phase as a film. The firstfluid forms the inner phase, the second fluid the outer phase. Themethod further includes providing at least one magnetically attractableparticle, which includes a ligand that is capable of binding targetmatter (supra). The method also includes disposing the at least onemagnetically attractable particle into the first fluid forming the innerphase of the fluid droplet. As indicated above, the at least onemagnetically attractable particle may be disposed into the first or thesecond fluid at any time during this method, since the fluid droplet isa self-organising system. A magnetic particle may for example bedisposed into the droplet, for example into the first fluid, before thedroplet of the first fluid is dispensed into the second fluid.

As mentioned above, the inner phase of the droplet may directly contactmatter that is included in at least one surface on which the droplet isor is intended to be disposed. Two illustrative examples of such matterare a solid surface or the surface of a fluid. The at least one surfacemay have any shape and geometry as long as it is of such a texture, e.g.roughness and waviness, that the fluid droplet remains intact upon beingcontacted therewith. As an illustrative example, it will typically berequired to provide a surface with a roughness for the fluid of theinner phase of the fluid droplet that is low enough to allow a fluiddroplet that gets in contact therewith to remain intact. The term“intact” refers to the existence of a defined droplet including twophases. The fluid droplet is thus understood to remain intact, while itis for instance spread to a desired extend, or merged with anotherdroplet. The at least one surface may for example be concave or convexrounded (cf. FIGS. 1D and 1E) or a combination thereof (FIG. 4C). In oneembodiment the at least one surface is essentially flat (cf. e.g. FIGS.1A-1C). In another embodiment the at least one surface has the form of acylinder, along the surface of which a droplet may be rotated.

The method of the present invention includes providing at least onesurface, such as e.g. described above. In some embodiments more than onesurface is provided, such as for instance two, three, four etc.surfaces. In one embodiment at least two surfaces are facing each other.The surface(s) may be of any material as long as it is of such awettability for the fluid of the inner phase of the fluid droplet thatthe fluid droplet remains intact upon being contacted therewith. In oneembodiment where more than one surface is provided, for instance twosurfaces facing each other, all respective surfaces are of such atexture and such a wettability for the fluid of the inner phase that thefluid droplet remains intact upon being contacted therewith.

Where for instance the inner phase of the fluid droplet is a polarliquid, such as an aqueous fluid, the at least one surface may benon-polar. In one embodiment the inner phase of the fluid droplet is anaqueous fluid, e.g. water, and the at least one surface is non-polar. Arespective non-polar surface may in some embodiments be selected fromthe group consisting of silicone (including surface-modified silicone),a polymer such as plastic (whether a biopolymer or a synthetic polymer,including a partially fluorinated polymer, a perfluorinated polymer, anda surface-modified polymer), surface-modified silicon oxide,surface-modified silicon hydride, surface-modified paper,surface-modified glass such as e.g. surface-modified pyrex,surface-modified quartz, surface-modified glimmer, surface-modifiedmetal, surface-modified alloy, surface-modified metal oxide,surface-modified ceramic, and any composite thereof. As a furtherillustrative example, the inner phase of the fluid droplet may behydrophilic and the at least one surface may be hydrophobic oroleophobic. As yet another illustrative example, the inner phase of thefluid droplet may be non-polar and the at least one surface may bepolar.

A surface modification is typically obtained by a treatment carried outto alter characteristics of a solid surface. Such a treatment mayinclude various means, such as physical, e.g. mechanical, thermal, orelectrical means, chemical means, or electrochemical means. As anexample, a surface of plastic materials can be rendered hydrophilic viatreatment with dilute sulfuric acid, chromic acid, a solution ofpotassium permanganate, or dilute nitric acid. As another example, apolydimethylsiloxane (PDMS) surface can be rendered hydrophilic by anoxidation with oxygen or air plasma. The surface of a hydrophobicpolymer, such as polymethylmethacrylate, polytetrafluoroethylene,polyethylene terephthalate, and polycarbonate, may also be renderedhydrophilic by means of ionic radiation in the presence of a reactivegas, as described by Kim et al (2003 ECI Conference on Heat ExchangerFouling and Cleaning: Fundamentals and Applications [2003], Vol. RP1,107-114). Silicon may be rendered hydrophilic by dipping inH₂O/H₂O₂/NH₄OH. Furthermore, the surface properties of any hydrophobicsurface can be rendered more hydrophilic by coating with hydrophilicself-assembled monolayers, a hydrophilic polymer or by treatment withsurfactants or plasma treatment with polymeric precursors.

Where a method according to the present invention is to be combined withanother method such as an analytical or preparative method (see alsobelow), it may be desired to provide a surface that allows, or isadvantageous for, carrying out both such a further method and a methodaccording to the present invention. During, or before, carrying out sucha further method the integrity of the two phases of the fluid dropletmay be affected or degraded. As a consequence matter that is located inthe inner phase of the fluid droplet may be exposed to another fluidphase and contact the surface. The availability of various suitableinner and outer phases for the fluid droplet used in the presentinvention typically allows for a flexible selection of a chemicalsurface treatment, including a coating. Therefore often the same surfacecan be used during both the method of the present invention and asubsequent method.

As an illustrative example, it may be desired to perform anelectrophoretic separation or an isoelectric focussing, for instance bysubjecting the magnetic particles, whether included in the fluid dropletused in the present invention or not, thereto. It may for instance bedesired to provide a surface with minimal interactions for any matterpresent, which is detectable by the selected method. Where it is forinstance desired to analyse the purity of an isolated protein byapplying an electromagnetic field (such as an electrophoretic method),analysis results may be falsified by a surface that significantlyinteracts with proteins. Two illustrative example of a suitable surfacecoating with minimal protein interactions are the polar polymerpoly-N-hydroxyethylacrylamide and poly(ethylene glycol)-terminatedalkyltrichlorosilane. It is likewise known that the properties of asurface of a device used for isoelectric focusing affect the efficiencyfor obtaining narrow isolated zones during both the focusing andmobilization processes. Examples of surface treatments that may be usedto achieve a high separation using a pH gradient in isoelectric focusinginclude, but are not limited to, a highly polar polymer coating such aspolyacrylamide, polyvinylpyrrolidone, polyethylene glycol, poly(vinyl)alcohol, or a fluorocarbon coating.

Examples of a chemical surface treatment include, but are not limited toexposure to, or reaction with, hexamethyldisilazane,2-hydroxy-4-(3-triethoxysilylpropoxy)diphenylketone,3-triethoxysilyl)propylsuccinic anhydride,2-[methoxy(poly-ethyleneoxy)propyl]trimethoxysilane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)triethoxysilane,(tridecafluoro-1,1,2,2-tetrahydrooctyl)triethoxysilane,(heptadecafluoro-1,1,2,2-tetrahydrodecyl)trichloro-silane,(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane,methacroyloxymethyltris(trimethylsiloxy)silane, PlusOne™ Repel-Silane ES(a 2% solution of dimethyldichlorosilane dissolved in octamethylcyclo-octasilane, GE Healthcare), SIGMACote° (a chlorinatedorganopolysiloxane in heptane), 3-mercaptopropyltrimethoxysilane,octadecylsilane, octadecyltrimethoxysilane,epoxypropoxypropyltrimethoxysilane,2-(diphenylphosphino)ethyltriethoxysilane,bis(2-hydroxyethyl)-3-aminopropyl-triethoxysilane,aminobutyltriethoxysilane, (3-acryloxypropyl)trimethoxysilane,trimethylchlorosilane, dimethyldichlorosilane, propyltrichlorosilane,tetraethoxysilane, glycidoxypropyltrimethoxysilane,3-aminopropyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-(2,3-epoxypropoxyl)propyltrimethoxysilane, polydimethylsiloxane (PDMS),γ-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, poly(methyl methacrylate)or a polymethacrylate co-polymer, urethane, polyurethane,fluoropolyacrylate, Teflon AF 1600, 2400, and 2200, poly(acrylic acid)(PAA), poly(methoxy polyethylene glycol methacrylate), poly(dimethylacrylamide), poly N-acroyl-aminoethoxyethanol (AAEE), polyacrylamidegrafted with benzophenone and Pluronic-F-68 (Li et al., Electrophoresis2005, 26, 1800-1806), poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA),α-phosphorylcholine-o-(N,N-diethyldithiocarbamyl)undecyloligoDMAAm-oligo-STblock co-oligomer (cf. e.g. Matsuda, T et al.,Biomaterials, (2003), 24, 4517-4527), poly(3,4-epoxy-1-butene),3,4-epoxy-cyclohexylmethylmethacrylate, 2,2-bis[4-(2,3-epoxy propoxy)phenyl]propane, 3,4-epoxy-cyclohexylmethylacrylate,(3′,4′-epoxycyclohexylmethyl)-3,4-epoxycyclohexyl carboxylate,di-(3,4-epoxycyclohexylmethyl)adipate, a copolymer of polyethyleneglycoland polypropyleneglycol such as UCON (Analabs, Norwalk, Conn., USA),bisphenol A (2,2-bis-(p-(2,3-epoxy propoxy) phenyl) propane),2,3-epoxy-1-propanol, polyvinylalcohol, polyvinyl pyrrolidone, dextran,surfactants such as dodecyldimethyl (3-sulfopropyl) ammonium hydroxide(Cl₂N₃SO₃), hexadecyldimethyl (3-sulfopropyl) ammonium hydroxide(C₁₆N₃SO₃), and coco (amidopropyl)hydroxyl dimethylsulfobetaine(RCONH(CH₂)₃N⁺(CH₃)₂CH₂CH(OH)CH₂SO₃ ⁻ with R=C₈-C₁₈), including apolymer surfactant such as e.g. Supelcoat PS2 (Supelco, Bellefonte, Pa.,USA), methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, orhydroxypropylmethylcellulose. A coating with a polymer may for instancebe carried out by means of chemical vapour deposition or bypolymerisation on the surface following an exposure of the surface to adifunctional reagent, as disclosed in US patent application2005/0249882. In the latter case a stable covalent linkage between thesurface and the polymer coating is generated. It may for example bedesired to select this technique in embodiments where the surface isrough or contains micro- or nanocavities.

Furthermore, the at least one surface may provide areas of differentsurface characteristics. In the above illustrative example of an innerphase of the fluid droplet being a polar (e.g. hydrophilic) liquid, someareas of the surface may for example be more non-polar (e.g.hydrophobic) than others, or some regions may be polar (e.g.hydrophilic). As an illustrative example, a surface area of increasedpolarity may be desired to achieve a spreading of a droplet on aDNA-array for hybridization. Any part of the at least one surface mayalso be treated in such a way that it provides respective polar ornon-polar surface characteristics. For example a solid surface may betreated respectively.

A common way of defining the wettability of a surface for a fluid suchas a liquid is the contact angle (also termed wetting angle) between adroplet of the fluid in thermal equilibrium on a horizontal surface,which is generally smooth and homogeneous, typically surrounded by a gassuch as air. In this respect, a person skilled in the art will be awareof the fact that an increasing roughness of a surface typicallyincreases the contact angle.

Depending on the type of surface and fluid, the droplet may have avariety of shapes as illustrated in FIG. 1. FIG. 1 also shows therespective contact angle θ of the inner phase of the depicted fluiddroplets. This contact angle is generally determined individually foreach respective phase of interest (cf. below), for instance the innerphase and the outer phase of a fluid droplet to be used in the presentinvention. In some embodiments the wettability of a surface for a fluiddroplet that includes an inner phase and an outer phase, may bedetermined in the same way, in particular where the outer phase is abulk phase. It is however more convenient to determine the contact angleof each phase separately. A contact angle θ is given by the anglebetween the interface of the droplet and the horizontal surface. Such acontact angle θ is a thermodynamic variable that depends on theinterfacial tensions of the surfaces involved. It reflects the balanceof forces exerted by an attraction of molecules within the droplet toeach other versus the attraction or repulsion those droplet moleculesexperience towards the surface molecules.

The most commonly used technique of determining the contact angle is theso called static or sessile drop method in a configuration of a singlephase, resembling the inner phase as shown in FIG. 1. The measurementusually involves a successive addition of fluid droplets until a plateauin the contact angle is reached. The value at a respective plateau iscalled the advancing contact angle. A further value that is regarded asless meaningful in this respect is the so called receding contact angle.It is obtained by continuing an advancing contact angle experiment byimmediately subsequently monitoring the contact angle as equivalentvolume droplets of fluid are successively retracted from the droplet.Further means of determining the contact angle include the WilhemlyPlate method, the Captive Air Bubble method, the Capillary Rise method,and the Tilted-drop measurement.

A contact angle θ of zero results in wetting, while a contact angle θbetween about 0 and about 90 results typically in spreading of the fluiddroplet, in particular at values in the range below about 45 degrees.Contact angles θ greater than about 90 indicate the fluid tends to beador shrink away from the solid surface (cf. FIG. 1C for an example). Asalready indicated above, the contact angle is typically determined for asingle phase of fluid. Accordingly the contact angle of the inner andthe outer phase of the fluid droplet are typically determinedseparately. In some embodiments the at least one surface used in thepresent invention is of a wettability for the fluid of the inner phaseof the fluid droplet that a fluid droplet of a single phase, consistingof the respective fluid, when disposed thereon can be characterized byan advancing contact angle θ of about 50 degrees or higher. Thus, thewettability of the surface is characterized by an advancing contactangle θ at the interface of a fluid droplet, which is made up of theinner phase of the above defined fluid droplet, with the at least onesurface of about 50 degrees or higher. In some embodiments the at leastone surface used in the present invention is of a wettability for thefluid of the outer phase of the fluid droplet that a fluid dropletconsisting of the fluid of the outer phase is characterized by anadvancing contact angle θ at the interface of a respective fluid dropletwith the at least one surface of about 50 degrees or higher. In someembodiments the at least one surface used in the present invention is ofa wettability for the fluid droplet as a whole that is so low that it ischaracterized by an advancing contact angle θ at the interface of saidfluid droplet with the at least one surface of about 50 degrees orhigher.

In some embodiments the surface, e.g. a solid surface, is furthermoreinert against the fluid of the inner or the outer phase of the fluiddroplet. Such embodiments allow for multiple reusing of the device. Anillustrative example of a material that is inert against most corrosivemedia is a fluoropolymer such as fluoroethylenepropylene (FEP),polytetrafluoroethylene (PFTE, Teflon), ethylene-tetrafluoroethylene(ETFE), tetrafluoroethylene-perfluoro-methylvinylether (MFA), vinylidenefluoride-hexafluoro-propylene copolymer,tetrafluoroethylene-hexafluoropropylene copolymer, vinylidenefluoride-hexafluoropropylene-tetrafluoroethylene terpolymer,perfluoromethyl vinyl ether-tetrafluoroethylen copolymer,perfluoroalkoxy copolymer (PFA), poly(vinyl fluoride),polychlorotrifluoroethylene, fluorosilicones, or fluorophosphazenes.

Additionally, and in particular where a respective phase is a liquidphase, the inner or the outer phase of the fluid droplet may include anionic or non-ionic surfactant, for example a perfluorocarbon-surfactant.Typically the surfactant adsorbs primarily at the solid-liquid andliquid-liquid and liquid-vapour interfaces near the contact line region,where applicable. As an illustrative example, it may be desired to use asurfactant in order to reduce nonspecific interactions of a sampleincluded in the inner phase of the fluid droplet with a surface.Numerous surfactants, which are partly hydrophilic and partlylipophilic, are used in the art, such as for instance alkyl benzenesulfonates, alkyl phenoxy polyethoxy ethanols, alkyl glucosides,secondary and tertiary amines such as diethanolamine, Tween, Triton 100and triethanolamine, or e.g. fluorosurfactants such as ZONYL® FSO-100(DuPont).

The method of the present invention also includes disposing the fluiddroplet onto the at least one surface. The fluid droplet may be disposedby any means. As an example, a dispenser may be provided. A dispensermay employ any suitable device or mechanism in order to provide anddispense a fluid droplet of a desired size. Examples include, but arenot limited to, piezoelectric pipettors, syringe pump-based pipettors,peristaltic pumps, touch-off dispensing, pressure-mediated dispensing,inkjet dispensing (including syringe-solenoid dispensing), pin-transfer(cf. Rose, D, Drug Discovery Today (1999), 4, 411-419 for a review).Disposing the fluid droplet may also rely on, or be assisted by, theproperties of the magnetically attractable matter included therein.Accordingly, a magnetic, electromagnetic, electrical or electrostaticfield may be used. As an illustrative example, a magnetic plug may beinjected from a capillary under the influence of a magnetic,electromagnetic or electrical field, for instance by dielectrophoresis.By means of the dispenser the fluid droplet may in one embodiment bedisposed onto the surface without contacting the same. In yet anotherembodiment the fluid droplet may be dispensed directly onto a surface bymeans of contact dispensing. Where desired, the dispensed quantities maybe measured, e.g. by means of a camera as disclosed in US patentapplication 2003/0209560.

FIGS. 7, 16 and 18 depict illustrative examples of fluid droplets, asused in the present invention, on a surface. The surface may before,during or after dispensing a fluid droplet thereon have any orientationrelative to the ground. In embodiments where an essentially flat surfaceis provided and the fluid droplet is handled in a gas such as air, itmay for convenience be desired to dispose the fluid droplet on top ofthe respective surface. Likewise, in such a case it may be desired tomaintain the respective surface in an essentially horizontal position inorder to assist controlling the position of the fluid droplet,especially where it is desired to position the fluid droplet my means ofa magnetic field and thereafter to terminate the magnetic field. In sucha case the fluid droplet is typically handled below or above therespective surface as depicted in FIG. 3A and FIG. 3B. In embodimentswhere a concave or convex surface is provided and the fluid droplet ishandled in a gas such as air, it may for instance for convenience bedesired to dispose the fluid droplet at a top or bottom position of adent, relative to the direction of action of gravitation.

Since the method of the present invention typically does not require anyfurther mechanical parts, it relies on a microfluidic system that isrobust. The method of the invention typically does neither requirevalves, so that no dead volume occurs. Accordingly the method of thepresent invention is well suited for processing sample volumes in thenanoliter scale and below (supra) without any material loss.

Typically the method of the invention involves controlling the positionof the fluid droplet, in particular relative to the at least onesurface. This position may for instance be controlled by geometricalmeans, such as a concave surface (cf. FIG. 1E). Another means ofcontrolling the position of the fluid droplet includes mechanical force.Such mechanical force can for instance be applied by contacting thefluid droplet with a further surface such as for instance the surface ofa pipette tip. A further means of controlling the position of the fluiddroplet includes the application of acoustic waves as disclosed byGuttenberg, Z. et al., Lab on a Chip (2005) 5, 308-317. Yet anothermeans of controlling the position of the fluid droplet includes theapplication of a thermal gradient, e.g. of the at least one surface. Arespective thermal gradient may for instance be obtained by means of anIR-laser.

In some embodiments the method of the invention controlling the positionof said fluid droplet relative to the at least one surface furtherincludes exposing the fluid droplet to a magnetic or an electromagneticfield. This exerts a force on the magnetic particles, such that thedroplet as a whole is forced to follow any movement of the magneticparticles. Thereby the position of the fluid droplet can be controlled.In some embodiments a constant magnetic or electromagnetic field isapplied, while in other embodiments the magnetic or electromagneticfield is altered during the method of the invention. In some embodimentscontrolling the position of the fluid droplet includes moving thesurface, for instance under a constant magnetic or electromagneticfield. As a consequence the position of the fluid droplet relative tothe surface can be altered. In some embodiments several means ofcontrolling the position of a fluid droplet may be combined (cf. alsobelow for further examples). In some embodiments the process is onlyperformed once the fluid droplet has been positioned by means of themagnetic or electromagnetic field. In one of these embodiments, themagnetic or electromagnetic field is terminated after the fluid droplethas been placed in a desired position. As an illustrative example, aregion of the at least one surface may be exposed to a condition such asan altered temperature, an (altered) magnetic field, an (altered)electrical field (including an electrostatic field), an (altered)electromagnetic field, an altered pressure, an (altered) wavelength, an(altered) frequency, an (altered) amplitude, an (altered) chemicalconcentration, and an (altered) chemical composition (such as a gasflow). In such a case controlling the position of the fluid droplet mayinclude moving the fluid droplet into the region of the at least onesurface that is being exposed to this condition.

In some embodiments of the method of the invention, exposing the dropletto an electric/magnetic field includes repelling the droplet. In some ofthese and in other embodiments exposing the droplet to anelectric/magnetic field includes attracting the droplet. Attracting orrepelling the droplet by means of a magnetic or an electric field mayfor instance be achieved by means of a bar magnet, an electromagnet, anarray of bar magnets or electromagnets, or any combination thereof. Arespective magnet may be moved in order to move one or more fluiddroplets. The attractive or repelling forces of one or more magnets ofan array may also be modified in order to control the position of afluid droplet.

In some embodiments where at least two surfaces as defined above areprovided, the fluid droplet may be transferred from one surface toanother and be moved between them. In such embodiments controlling theposition of the fluid droplet may include moving the fluid dropletbetween two surfaces by means of a magnetic or an electromagnetic field.

The method of the invention further includes performing a process on thebiological and/or chemical sample in the fluid droplet. Any process maybe performed that can be performed in a fluid droplet. Examples ofprocesses that may be performed include, but are not limited to, aphysical detection of target matter suspected or known to be included inthe sample, a chemical reaction, a cell lysis, an extraction of amolecule from an organism or a part of an organism, a release of amolecule from an organism, and any combination thereof. Examples of aphysical detection include, but are not limited to, a spectroscopic, aphotochemical, a photometric, a fluorometric, a radiological, anacoustical, an electrochemical, a colourimetrical, a diffractional, aninterferometrical, an elipsometrical, and a thermodynamic detection andinclude for instance the use of photoactive, fluorescent, radioactive orenzymatic labels. Two illustrative examples of a spectroscopic methodare Raman microscopy and coherent anti-Stokes Raman scattering (CARS)microscopy. The latter technique is for example suitable for selectiveimaging of specific molecules of interest. Examples of a chemicalreaction include, but are not limited to, a chemical synthesis, achemical degradation, an enzymatic synthesis, an enzymatic degradation,a chemical modification, an enzymatic modification, an interaction witha binding molecule, and any combination thereof. Examples of anenzymatic synthesis include, but are not limited to a protein synthesis,a nucleic acid synthesis, a peptide synthesis, a synthesis of apharmaceutical compound, and any combination thereof. The method of theinvention is for example compatible with any biochemical transformationor assay format, e.g. the yeast-two-hybride system, small interferingRNA (siRNA), transfection, ligation, etc.

Performing a process may include an exposure to energy, for instance fora process to be initiated or catalyzed. Examples of energy that may beapplied, include, but are not limited to, infrared radiation, microwaveradiation, or photolytic energy. As an illustrative example, the surfacetogether with a fluid droplet may be placed biochip on a thin filmcooler/heater. Accordingly, chemical synthesis driven by elevatedtemperatures or requiring reduced temperatures may for instance beperformed by adjusting the temperature of the environment of the dropletrespectively. As another example, temperature-controlled biochemicalreactions between 4 and 100° C. can be performed. Thus,temperature-sensitive biological samples may for instance be stored.Further examples include, but are not limited to, cell isolation (cf.FIG. 9), cell incubation, cell lysis (FIG. 6), reverse transcription,polymerase chain reaction (FIG. 10, 12), and pyrosequencing (FIG. 13).Pyrosequencing is a real-time nucleic acid sequencing technique, whichis based on the detection of released pyrophosphate during the nucleicacid polymerization reaction (for an overview cf. e.g. Ronaghi, M,Genome Research (2001), 11, 3-11). Furthermore, the implementation ofdiverse optical detection systems, e.g. photodiodes (PD),photomultipliers (PMT), photon counting modules (PCM), spectrometers,and charge-coupled devices (CCDs) allows monitoring these biochemicalreactions in parallel and real-time.

As an illustrative example, a pathogen, a bacterium, a virus, or a DNAsequence may be detected using the present invention for identifying adisease state. Diseases which can be detected include, but are notlimited to, communicable diseases such as Severe Acute RespiratorySyndrome (SARS), Hepatitis A, B and C, HIV/AIDS, Dengue, swine fever,mouth-and-foot-disease, avian flu, anthrax, salmonella, malaria, polio,tuberculosis and influenza; congenital conditions that can be detectedpre-natally (e.g. via the detection of chromosomal abnormalities) suchas sickle cell anaemia, heart malformations such as atrial septaldefect, supravalvular aortic stenosis, cardiomyopathy, Down's syndrome,clubfoot, polydactyl), syndactyl), atropic fingers, lobster claw handsand feet, etc. The present method is also suitable for the detection andscreening for cancer, for identifying the pedigree of an animal, e.g. bymeans of a DNA-tag, or the detection and analysis of substances inblood, e.g. doping.

In other embodiments the method of the present invention may be employedfor the detection, reaction (including a binding reaction to abiological cell or a part thereof), synthesis, or any combinationthereof, of one or more pharmaceutical compounds, such as drugs. Asynthesis of a compound, such as a pharmaceutical compound, may forexample be performed as a solid-phase reaction on derivatised beads.Pharmaceutical compounds may for example be used in form of a library.Examples of such libraries are collections of various small organicmolecules, chemically synthesized as model compounds, or nucleic acidmolecules containing a large number of sequence variants. As an example,each compound of such a library may be disposed into one droplet. Suchdroplets may be provided in an automated way by commercially availablemachines, which are well known to those skilled in the art. The methodof the invention may for instance be used for drug screening or fordetermining the presence of a drug in a urine or blood sample.

As a further example, a cell culture media may be suspected to becontaminated (supra). In this case it may be desired to identify thetype of contaminant and to use the device of the invention for thispurpose. The magnetically attractable matter may in such embodiments forinstance be magnetically attractable particles carrying a ligand with anaffinity to the contaminant or with an affinity to other matter that hasan affinity to the contaminant.

In embodiments where it is desired to remove matter, such as by-productsor undesired matter of the sample, the process may be a washing processor a process including a washing step. It may also include splitting thefluid droplet into at least two daughter fluid droplets. As anillustrative example, a nucleic acid may be extracted from a cell and bebound by a ligand attached to magnetic particles, while cell debris andreagents are to be discarded. FIG. 4A illustrates an example of awashing step of a fluid droplet using a further, additional fluiddroplet. This further fluid droplet may also include two or more fluidphases. It may be provided on the same surface as the fluid droplet thatincludes two phases, magnetic matter and the sample, or it may beprovided on another surface (FIG. 4B). It is moved toward the fluiddroplet that includes two phases, magnetic matter and the sample (FIG.4A(1)). The arrow in FIG. 4A indicates the current position of apermanent magnet. The two fluid droplets merge (FIG. 4A(2)) and form onelarger fluid droplet (FIG. 4A(3)). To ensure a complete mixing andwashing a weak magnetic force may be applied that is sufficient to forinstance lift the magnetic particles within the droplet without raisingthe entire fluid droplet. By further moving the magnetic particles toone side (FIG. 4A(4)) a splitting of the droplet is initiated (FIG.4A(5)). The ratio of magnetic particles/outer phase, the volume ratio ofinteracting fluid droplets, their biochemical composition, the surfacemorphology, the surface chemistry, and the strength of the(electro)magnetic field gradient dictate whether the corresponding fluiddroplets move, merge, are ‘washed’ or split. During these manipulationsthe dead volume is zero, i.e. no material is lost even if nanolitervolumes are processed. Where desired, further functional units mayeasily be implemented in the method of the invention, e.g.piezoelectric-based actuators to assist or achieve mixing.

The inner phase of the droplet may be washed or exchanged with any fluid(see e.g. FIG. 18), for instance a solvent, an acid or a base, as longas the fluid allows for (a) the inner phase to remain essentially intactand (b) the magnetic particles to remain attractable to a magnet. Inembodiments where the outer phase forms a film surrounding the innerphase (supra), it may furthermore be desired to keep the outer phaseintact as a film. In embodiments where a ligand attached to magneticparticles is used to bind target matter, it may furthermore be desiredthat such a fluid allows for the ligand to remain intact and to bind thedesired target matter. At any point in time before, during or afterperforming a process, a mixing of the fluid droplet may be carried out,for instance by exposing the fluid droplet to ultrasound. Since thedroplet is based on a self-organising system, such mixing does notaffect the integrity of the droplet, but rather assists in achieving anequal distribution of matter within a phase within the droplet.

The possibility to perform transfers of matter such as washing allow forcomplex processes to be performed. Since desired target matter may bebound to ligands immobilized on magnetic particles, the possibility toadd, remove or exchange fluid, e.g. liquid, enables the isolation of anymatter, e.g. peptides, proteins, DNA, RNA, small organic molecules,metal ions, etc. (supra) at any desired stage or step, and complexbiochemical transformations can be carried out in sequence (FIG. 4).Furthermore the volume of the fluid droplet can be changed by severalorders of magnitude. Accordingly the method of the present inventionprovides an interface between the macroscopic and microscopic worldwithout any break in technology.

Where desired, the concentration of target matter in a sample may beincreased by volume reduction according to techniques well known in theart such as gel-filtration, ultrafiltration or dialysis. A low volume asused in the method of the present invention, in particular incombination with a high concentration of target matter, makes biosensingof biomolecules in low absolute numbers possible.

As a further illustrative, but not limiting example, the method of thepresent invention may be used to carry out a sandwich-type enzyme-linkedimmunosorbent (ELISA) assay. The uses and capabilities of this assay arewell known to those skilled in the art. By combining several fluiddroplets, wherein each droplet contains at least one or more of therequisite reagents for the binding, washing and detection steps, it ispossible to assay for the presence of target matter in a sample. Asdescribed above, the capture reagent may be coupled to magnetic beads.In a specific embodiment, the capture reagent is an antibody targeted toan antigen of interest. More specifically, the antibody may be directedto an antigen present in a HIV virus and the sample blood or serumsuspected to contain this HIV virus. Detection of an antigen can bemonitored through calorimetric and or spectroscopic analysis. This canoccur after elution of substrates captured onto the probe or within thedevice itself without recourse to an elution step.

FIG. 6 illustrates the use of the method of the present invention for apolymerase chain reaction. Temperature control can be achieved by meansof thin film heaters and temperature sensors. Module 1 represents amatrix of superparamagnetic particles, which are modified with ligands.These ligands are receptors directed against different cell surfacemarkers. Any cell of interest may in this way be isolated from bodyfluids or tissue as described above. A drop of capillary whole humanblood may be obtained by finger pricking with a lancet. This drop ofblood is placed onto module 2. Leucocytes may then be isolated accordingto the binding of their cell surface markers to the ligand immobilizedon the magnetic particles (cf. above, see also FIG. 5 and FIG. 7). FIG.8 illustrates by way of magnification an example of leucocytes bound toligands immobilized on magnetic particles. Leucocytes can be thermallylysed on one of the four thin film heaters of module 3. The polymerasechain reaction (PCR) is performed in a clock-wise manner by guiding thesample over three different temperature zones (cf. module 3 in FIG. 6).FIG. 17 depicts a temperature profile measured using the method of thepresent invention. FIG. 11 verifies that a PCR product obtained by themethod of the present invention is of a quality that does not differfrom a product obtained by conventional methods used in the art. Usingtime-space conversion makes multiplexing of samples possible. Abiotinylated PCR product may be generated, which can be bound tostreptavidin coated superparamagnetic particles. The amplificationproduct can be chemically denaturated on module 4 and annealed to asequencing primer for pyrosequencing (PSQ), which may be carried out ina clock-wise manner on module 5 by moving the sample through fourdifferent VRCs containing the bases G, A, T and C. FIG. 13 shows anexample of a respective analysis. Using time-space conversion makesmultiplexing of samples possible. If desired, the samples can be storedon module 6 after pyrosequencing, again in a matrix-like format.Alternatively they can also be processed further.

A further example of performing a process on the biological and/orchemical sample in a fluid droplet is mixing the interior of the fluiddroplet. The term “mixing” refers to passive interblending, i.e. bydiffusion, to active mixing by means of e.g. applying external energy orforces, as well as to combinations thereof. Active mixing within astationary or moving droplet may for example include agitating thesuperparamagnetic particles. Such agitation may be achieved for instanceby altering a magnetic or electromagnetic field to which the droplet isexposed. It may also be achieved by altering the relative position of adroplet in a constant magnetic or electromagnetic field by moving therespective surface (supra). Yet another means of active mixing relies onultrasound, which is illustrated in the appending Examples.

Yet another example of performing a process on the biological and/orchemical sample in a fluid droplet is filtering the fluid dropletthrough another fluid droplet, as illustrated in FIG. 18. Such afiltration is typically performed by means of moving a smaller dropletcontaining functionalized superparamagnetic particles with immobilizedtarget matter through a bigger fluid droplet. In this way undesiredcomponents such as for example by-products, impurities, substrates,reagents, solvents or solvent components, salts, enzymes, waste, orbuffers, can be diluted in the bigger droplet. Upon further movement ofthe magnetic particles out of the bigger droplet, essentially only thesuperparamagnetic particles including the immobilized target matter arebeing removed from the bigger droplet, while most of the undesiredmatter is being left behind. Due to the self-organizing nature of thesystem, the outer phase or a part thereof, is likewise removed from thelarger droplet. In case of an outer phase in form of a film, a thin filmof the outer phase may for example surround a small remaining amount ofinner phase. In this way it is possible to substantially remove matterfrom the fluid droplet that is not immobilized by the magnetic beads.(cf. the Examples and FIG. 20). The underlying purification effectresembles the mechanism known from affinity chromatography, where targetmatter is held back by functionalized column material forming thestationary phase, and rinsed/washed several times with a washingsolution, forming the mobile phase. In contrast to affinitychromatography, in the method of the present invention the washingsolution is the stationary phase, while the functionalized material isthe mobile phase. It should furthermore be noted that no dead volumeoccurs using the method of the present invention. Furthermore, incontrast to affinity chromatography, the method of the present inventionallows for the elution of target matter in nanoliter volumes. Thisadvantage is crucial in applications such as biosensing, when forexample a high concentration of target matter is present in tinyvolumes, or where fast kinetics are to be analysed.

Any part of the method of the present invention may be performed in amanual or in an automated way. Automated distribution of compounds,fluid and reagents, automated incubators and high-performancefluorescence readers, including plate readers, are already wellestablished in the art. Typically, such equipment can directly be usedwith an apparatus of the present invention. Where required, adaptationsof either such equipment or of the apparatus of the invention to aparticular application are easily performed by a person skilled in theart.

Real time detection may provide an amplification plot depicting thefluorescence signal versus reaction time expressed as cycle numbers (seeFIG. 16). An increase in fluorescence above the baseline indicates thedetection of accumulating amplification product. Where a fixedfluorescence threshold is set above the baseline, the fluorescencesignal thus passes this threshold at a certain time point. As time isexpressed in terms of cycle numbers, a so called cycle threshold number(or value) or Ct value is obtained. The smaller this number, the furtherto the left is a respective fluorescence curve located in theamplification plot and the faster does amplification occur. The higherthis number, the slower an amplification occurs and the less it becomesdistinguishable from non-specific background reactions. An illustrationof obtained fluorescence signals using the method of the presentinvention and is depicted in FIG. 10.

The method of the invention may be combined with such analytical andpreparative methods, as for instance surface plasmon resonance, resonantmirror, reflectometric interference, giant magneto resistance, massspectroscopy, ellipsometry, isoelectric focusing, chromatographymethods, electrochromatographic, electrokinetic chromatography andelectrophoretic methods. Examples of electrophoretic methods are forinstance Free Flow Electrophoresis (FFE), pulsed field gelelectrophoresis, Polyacrylamide gel electrophoresis (PAGE), CapillaryZone or Capillary Gel Electrophoresis. Surface immobilization ofmagnetic particles by charged proteins is for example known to shifttheir electrophoretic mobility up to several-fold. The combination withsuch methods may include a common step or a common device. As anexample, a separation of proteins may be performed on a micro chip, forinstance by isoelectric focussing. Subsequently a sample of theseparation medium, e.g. a solution of ampholytes in water, known orsuspected to contain matter, such as a protein, of interest may be usedas e.g. the inner phase of a fluid droplet of the present invention. Incase of the separation medium being a gel, the matter of interest mayneed to be extracted. The variety of suitable fluids for the inner andouter phase of the fluid droplet used in the present invention usuallyallows for the selection of a surface material that is well suited forusage as a surface for isoelectric focussing. As a consequence, a commonsurface may be shared for both methods where desired. Examples of achromatography method include for instance gel filtration, sizeexclusion chromatography, ion exchange chromatography, affinitychromatography, hydrophobic interaction chromatography or hydrophobiccharge induction chromatography. As an illustrative example, arespective analytical or preparative method may be performed before orafter processing a sample using the method of the present invention.

In order that the invention may be readily understood and put intopractical effect, particular embodiments will now be described by way ofthe following non-limiting examples.

EXAMPLES Proplet Manipulation

An in-house built array of electromagnets, a permanent M1219-X neodymiumiron boron disc magnet (Assemtech), fixed to the handle of an analoguealarm-clock (Citizen) by double-sided tape (3M) (cf. FIG. 18 a), or an AProScanII-motorized stage system (Prior), which was moved relatively toa stationary permanent neodymium iron boron disc magnet (Farnell) andcontrolled by a joystick or LabVIEW 8-software (National Instruments)was used to manipulate fluid droplets containing superparamagneticparticles (cf. FIG. 19).

The fluid droplets contained a polar inner liquid phase and a non-polarouter liquid phase. The inner phase was aqueous and contained buffer,reagent(s) and superparamagnetic particles (cf. below). Ultrafiltratedmineral oil (Fluka) was used as immiscible liquid forming the outerphase in form of a thin film surrounding the inner phase. Forapplications at room temperature (rt), the volume ratio of the aqueousphase to the immiscible phase was 10:1, whereas for applicationsrequiring elevated temperatures, e.g. pyrosequencing (PSQ), reversetranscription (RT), cell lysis, and polymerase chain reaction (PCR), itwas 1:5.

The microfluidic manipulations included merging, mixing,washing/filtering, and splitting of droplets. Mixing within the dropletwas achieved either by means of agitation of the superparamagneticparticles by altering the magnetic/electromagnetic field or by acousticcavitation/streaming by ultrasound. In some cases both mixing techniqueswere combined.

To generate ultrasound, a 33250A 80 MHz function/arbitrary waveformgenerator (Agilent), equipped with an in-house built amplifier, and alead zirconium titanate (PZT) disc (Phillips), glued to the substrate byan epoxy adhesive (Alteco), were used. Depending on the experiment,different waveforms in the kHz-range provided fast and efficient mixing.Except for the PCR solution, all droplets, i.e. blood, functionalizedsuperparamagnetic particles, mineral oil, capping solutions, washingsolutions, substrates, etc., were disposed onto the surface at thebeginning of the experiment and the droplet containing thesuperparamagnetic particles was successively merged, mixed,washed/filtered, and split with these droplets.

FIG. 18 shows moving, merging, washing/filtering, and splitting for thefollowing example of the isolation of white blood cells from fresh,capillary whole human blood using functionalized superparamagneticparticles (see below). Since white blood cells express CD15 and CD45antigens (cell surface markers) on their cell membrane, they can beselectively removed by immunoreaction with anti-CD15 and anti-CD45antibody-coated superparamagnetic particles. FIG. 18A: a permanentM1219-1 neodymium iron boron disc magnet (Assemtech), fixed on thehandle of an analog clock (Citizen) by double-sided tape (3M), is usedto move, merge, wash/filter, and split droplet(s) containingfunctionalized superparamagnetic particles (1), which are placed on topof a Teflon AF (DuPont)-coated glass slide (24×60 mm, thickness 180 μm,vfm CoverSlips, CellPath) above the magnet; for a better visibility inthe following figures, a white paper is placed between the clock and theglass substrate. FIG. 18B: A droplet containing 100 nl of Dynabeads®CD15 and CD45 (Dynal Biotech) (26) is moved towards a droplet containing100 nl fresh, capillary whole human blood (27), and merged (FIG. 18C,18D). After merging of the two droplets, the combined droplet (28) ismoved towards a droplet containing 10 μl of washing solution (0.01MPBS/0.1% BSA), (29) (FIG. 18E, FIG. 18F); during this incubation step,the white blood cells are immobilized on top of the functionalizedparticles, whereby the agitation of the moving superparamagneticparticles supports active mixing. In addition, acoustic streaming byultrasound can be used to shorten the incubation time for this purpose.It should be noted that it is for a number of applications desirable toavoid acoustic cavitation at higher amplitudes, as this mode leads tothe rupture/lysis of white blood cells. After merging (FIG. 18G), theimmobilized white blood cells are washed/filtered. The term‘washing’/‘filtering’ refers to moving the functionalizedsuperparamagnetic particles with immobilized white blood cells through abigger droplet containing the washing solution (FIG. 18H, FIG. 18I).Upon doing so only the superparamagnetic particles including theimmobilized white blood cells, together with a thin film of immiscibleliquid of the outer phase by self-organization, leave (FIG. 18J) thebigger droplet. All other matter that is included in the bigger droplet,such as impurities, e.g. red blood cells, buffer, salts, EDTA (servingas anti-coagulant), heparin, RNases, DNases, etc. remain in the biggerdroplet. The presence of red blood cells turns the washing solution redin color (cf. the dark stain of the remaining droplet). At the same timeimpurities, eventually part of the slurry of functionalizedsuperparamagnetic particles with immobilized white blood cells, leavingthe bigger droplet are being diluted. Thereby, the dilution factor isdependent on the volume ratio of the interacting droplets: if forinstance a droplet of 0.1 μl of functionalized superparamagneticparticles with immobilized white blood cells is washed three times by 10μl of washing solution, the remaining impurities within the slurry arediluted one million-fold [(1:100)³]). Thereafter, the functionalizedsuperparamagnetic particles with immobilized white blood cells leave thewashing solution (29) (FIG. 18J) and are moved towards a second washingsolution (30) (FIG. 18K, FIG. 18L) for a second washing step. To enhancewashing, the functionalized superparamagnetic particles with immobilizedwhite are moved for and backwards several times within the washingsolution (FIG. 18M-FIG. 18U), before they leave the washing solution(FIG. 18V, FIG. 18W). Finally, the purified white bloods cells are readyfor downstream applications, e.g. RT-PCR. The filter-efficiency of onewashing solution for removing red blood cells (the main component ofblood, which interferes with downstream applications due tocontamination with RNases and DNases), was estimated by counting the redblood cells using a Neubauer-hemacytometer (FIG. 20A, FIG. 20B) and is˜100 000-fold, i.e. the combination of two washing solutions of 10 μleach removes all red blood cells within a sample volume of up to 1 ml(absolute number of red blood cells in a 1 ml sample is 5×10⁹).

Thermal Management

The temperature control module, fabricated in house, comprised platinum(Pt) thin film heaters and temperature sensors, and an applicationspecific integrated circuit (ASIC) controller (cf. FIG. 17).Alternatively, PCR experiments (without optical detection) were carriedout on a PCT-200® Peltier Thermal Cycler (MJ Research), equipped with aSlide Griddle™ adaptor (MJ Research). The set-up of the thermalmanagement is similar to other devices used in the art, such as e.g.disclosed by Guttenberg, Z. et al. (supra). Commercially availablecustom-made PCR-chips including controller/software were found suitablefor usage in the method of the present invention. In some tests PCRchips from Advalytix (Brunnthal, Germany, www.advalytix.com) and of theInstitute for Physical High Technology e.V. (Jena, Germany) were used.

Optical Detection

Fluorescence was detected by an Axiotech vario fluorescence microscope(Zeiss), equipped with a X-Cite 120 fluorescence illumination system(EXFO Life Sciences), a HE 38 FITC filter set (Zeiss), a 5784-20photomultiplier tube (Hamamatsu) and recorded by a TDS50054B digitalphosphor oscilloscope (Tektronix) according to the manufacturer'sinstructions.

For bioluminescence detection, a H7421-40 photon counting module(Hamamatsu) was used instead.

Surface Modification

Hydrophobic as well as oleophobic surfaces were obtained by eitherchemical vapour deposition (CVD) of glass (Schott) or silicon (SiliconSense) substrates with(heptadecafluoro-1,1,2,2-tetrahydrodecyl)triethoxysilane (Gelest) in a15E oven (Yield Engineering Systems) at 150° C. and 0.5 mbar for 2 h, orspin-coating of glass, silicon or polymeric substrates (GeneralElectric) with a 1% solution of Teflon AF (DuPont) in FC-70 (3M). Staticcontact angles with water and mineral oil (Fluka), measured by an OCA 30contact angle measuring device (DataPysiscs), were >110 and >70°,respectively.

The following sequence, isolation of white blood cells (WBCs) from wholehuman blood, cell lysis of WBCs, PCR, and PSQ was performedsuccessively, without any user interference (FIG. 6).

Isolation of White Blood Cells

A droplet containing 0.1 μl of a 1:1 suspension of 200 μg μl⁻¹ ofDynabeads CD15 and CD45 (Dynal Biotech) in 0.01 M phosphate bufferedsaline PBS (Sigma-Aldrich)/1% bovine serum albumin (BSA) (Roth) wasmerged with a droplet containing 15 μl of fresh capillary whole humanblood, mixed, incubated at rt for 10 min, and washed successively in twodroplets containing 10 μl 0.01 M PBS/1% BSA and one droplet containing10 μl of the PCR mixture (FIG. 7). The white bloods cells (WBCs),attached onto the superparamagnetic particles, and were now ready forthe cell lysis.

To count the number of isolated WBCs, the droplet containing thesuperparamagnetic particles was merged (cf. below) with a dropletcontaining 10 μl of a 4% solution of paraformaldehyde (Roth) in 0.01 MPBS/1% BSA, mixed (cf. below), and incubated at 4° C. for 30 min, andwashed successively in three droplets containing 10 μl 0.01 M PBS/1%BSA. After fixation of the WBCs, the droplet containing thesuperparamagnetic particles was merged with a droplet containing 1 μl ofVECTASHIELD® mounting medium with 4′,6-diamidino-2-phenylindole (DAPI)(Vector) mixed, and incubated at 4° C. overnight. Finally, theDAPI-stained WBCs were covered with a high resolution-printed polymericfoil (Infinite Graphics) with a grid size of 500×500 μm, placed under aBX51 system microscope (Olympus), photographed with a DP70 digitalcamera (Olympus), converted into greyscale (FIG. 8) and countedsoftware-assisted by MetaMorph_V6.1 (Molecular Devices) (FIG. 9).

Cell Lysis

After isolation of the WBCs, the droplet containing thesuperparamagnetic particles was merged with a droplet containing 1 μl ofthe PCR mixture, mixed and incubated at 95° C. for 5 min. After thermallysis of the WBCs, the superparamagnetic particles were removed from thedroplet containing the PCR mixture. The released genomic DNA was nowready for the PCR.

PCR

A housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used as the target, whereby the amplicon length was 208 bp (MaximBiotech). Biotin-CTC ATT TCC TGG TAT GAC AAC GA (SEQ ID NO: 1) and GTCTAC ATG GCA ACT GTG AGG AG (Research Biolabs, SEQ ID NO: 2) were used asforward and reverse primers, respectively. The singleplex PCR mixturewas prepared based on the Taq PCR Core Kit (QIAGEN) in a 50 μl stocksolution and had the following composition: 23.0 μl diethylpyrocarbonate (DEPC) treated H₂O (Invitrogen), 10.0 μl 5× Q-solution,5.0 μl 5×PCR buffer, 5.0 μl 10% BSA, 1.0 μl 10 mM dNTPs, 0.5 μl 1:100SYBR Green (Invitrogen), 2.50 μl 10 μM forward and reverse primer each,and 0.5 μl 5 u μl⁻¹ Taq DNA polymerase. For this particular PCR, ˜1400WBCs, isolated as described above, were used as the template.

Thermocycling conditions were as follows: 45 cycles at 95° C. for 1 min,58° C. for 1 min, 72° C. for 1 min, and 80° C. for 15 s. The mean valueof the fluorescence intensity during this 80° C.-interval was extractedto follow the PCR in real-time (FIG. 10). For melting curve analysis,the sample was cooled down to 65° C. for 1 min, after which thetemperature was continuously raised to 95° C. with a slope of 0.01 K s⁻(FIG. 11). Finally, the PCR products were analyzed by capillaryelectrophoresis using a Bioanalyzer 2100 (Agilent Technologies) (FIG.12). After completion of the PCR, the biotinylated PCR product was readyfor PSQ.

PSQ

For the PSQ, the 5×96 PSQ™ 96 MA Pyro Gold Reagent Kit includingconsumables (Biotage) was used. CAT GGC AAC TGT GAG GAG (SEQ ID NO: 3)served as sequencing primer. A droplet containing 1 μl of a suspensionof 300 μg μl⁻¹ of Dynabeads® MyOne™ Streptavidin in 2× binding bufferwas merged (supra) with the droplet containing the PCR mixture, mixed,and incubated at 65° C. for 15 min. After immobilization of thebiotinylated PCR product, the droplet containing the superparamagneticparticles was merged with a droplet containing 10 μl of 1× denaturationsolution, mixed, incubated at rt for 1 min, and washed successively intwo droplets containing 10 μl of 1× washing buffer. Alternatively, thedouble stranded (ds) DNA could be denaturated thermally at 95° C. for 1min. After denaturation of the ds DNA, the droplet containing thesuperparamagnetic particles was merged with a droplet containing 1 μl ofa solution of 0.3 μM sequencing primer in 1× annealing buffer, mixed,incubated at 80° C. for 2 min, and cooled down to rt. The singlestranded (ss) DNA, attached onto superparamagnetic particles, was nowready for PSQ.

To verify the immobilization of ss DNA onto the superparamagneticparticles, the droplet containing the superparamagnetic particles wassuspended in 50 μl 1× annealing buffer and sequenced in a commercialPSQ™ 96MA system (Biotage) (FIG. 13).

Finally, the droplet containing the superparamagnetic particles wasmerged with a droplet containing 2.5 μl of a solution of dGTP, mixed,and then merged with droplet containing 10 μl of a 1:1 solution ofenzyme and substrate mixture (FIG. 14).

Affinity Purification of a Protein Green Fluorescent Protein

This example illustrates how a protein can be purified using the methodof the present invention.

A fluid droplet containing 1 μl of an aqueous inner phase containing 50mM Tris-HCl pH 7.7, 200 mM NaCl, 5 mM EDTA and Streptavidin-modifiedmagnetic agarose beads (QIAGEN, equivalent to a 4% (w/v) suspension),and an outer phase of ultrafiltrated mineral oil (Fluka) can be preparedafter washing the tagged agarose beads with 0.01 M phosphate-bufferedsaline (PBS). A droplet containing 1 μl of a crude bacterial lysate (E.coli) containing biotinylated recombinant green fluorescent protein(GFP) in above buffer (50 mM Tris-HCl pH 7.7, 200 mM NaCl, 5 mM EDTA)can be merged with the droplet containing the magnetic agarose beads.The obtained droplet can be incubated at room temperature for 45 min.Thereafter, the droplet can be washed successively in three dropletscontaining 10 μl of above buffer (50 mM Tris-HCl pH 7.7, 200 mM NaCl, 5mM EDTA). Elution of the purified GFP in the obtained droplet of 1 μlcan be performed by merging with a droplet of 25 μl containing 10 mMbiotin in above buffer, and mixing by exposure to ultrasound, whereafterthe magnetic agarose beads were removed. Quantification may be performedby any standard method such as the ratio of UV absorption at 280 and 260nm according to Layne, by performing a colour reaction in a referencedroplet, for example according to Bradford, separation bySDS-polyacrylamide-gel-electrophoresis and a subsequent stain, or byfluorescence detection using a reference solution of GFP of knownconcentration.

Enzyme-Linked Immunosorbent Assay (ELISA)

A droplet containing 1 μl of a suspension of carboxy-functionalizedsuperparamagnetic particles in 0.1 M 2-morpholinoethanesulfonic acid(MES) (Lancaster) was merged (supra) with a droplet containing 10 μl ofa 0.2 M solution of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride (Lancaster) and 0.02 M N-hydroxysuccinimide (NHS)(AlfaAesar) in 0.1 M MES, mixed, incubated at rt for 1 min, and washedin one droplet containing 10 μl 0.01 M phosphate buffered saline (PBS)(Sigma-Aldrich). After activation of the carboxy groups, the dropletcontaining the superparamagnetic particles was merged with a dropletcontaining 1 μl of a 1 μg μl⁻¹ solution of fluorescein (FITC)-labelledgoat anti-mouse IgG (whole molecule) (Gt×Ms IgG FITC) (Sigma-Aldrich) in0.01 M PBS, mixed, incubated at rt for 1 min, and washed in one dropletcontaining 10 μl 0.01 M PBS. After coupling of the Gt×Ms IgG FITC (FIG.15), the droplet containing the superparamagnetic particles was mergedwith a droplet containing 10 μl 0.1 M 2-aminoethanol (Sigma-Aldrich),mixed, incubated at rt for 1 min, and washed successively in threedroplets containing 10 μl 0.01 M PBS/1% BSA. After capping of theresidual succinimide ester groups, the droplet containing thesuperparamagnetic particles was merged with a droplet containing 1 μl ofa 1 μg μl⁻¹ solution of horseradish peroxidise (HRP)-labelled rabbitanti-goat IgG Fc (Rbt×Gt IgG Fc HRP) (Chemicon) in 0.01 M PBS/1% BSA,mixed, incubated at rt for 1 min, and washed successively in fivedroplets containing 10 μl 0.01 M PBS/1% BSA or 10 μl 0.01 M PBS/0.05%Tween 20 (Sigma-Aldrich). After immunoreacting of Gt×Ms IgG FITC andRbt×Gt IgG Fc HRP, the droplet containing the superparamagneticparticles was merged with a droplet containing 10 μl of a 1:1 solutionof peroxidase substrate solution A and B (Bethyl), mixed, incubated for1-10 min, and split (cf. FIG. 16).

1.-49. (canceled)
 50. A method of processing a biological and/orchemical sample, comprising: providing a fluid droplet, said fluiddroplet comprising an inner phase and an outer phase, wherein the outerphase is immiscible with the inner phase, and the outer phase issurrounding the inner phase as a film, and wherein the inner phasecomprises said biological and/or chemical sample, and the inner phase isshielded from the environment by the outer phase, and wherein said fluiddroplet comprises magnetically attractable matter; providing at leastone surface, the surface being of such a texture and such a wettabilityfor the fluid of said inner phase of the fluid droplet, that the fluiddroplet remains intact upon being contacted therewith; disposing saidfluid droplet onto said at least one surface; exposing said liquiddroplet to a magnetic or an electromagnetic field, thereby controllingthe position of said liquid droplet relative to said at least onesurface; and performing a process on the biological and/or chemicalsample in said fluid droplet.
 51. The method of claim 50, wherein thefluid of the inner phase of said fluid droplet is a liquid.
 52. Themethod of claim 51, wherein the fluid of the inner phase of said fluiddroplet is a liquid and the fluid of the outer phase of said fluiddroplet is a liquid.
 53. The method of claim 50, wherein themagnetically attractable matter is selected from the group consisting ofat least one magnetically attractable particle, a magnetic fluid, aniron-rich bacterium, and a combination thereof.
 54. The method of claim50, wherein controlling the position of said fluid droplet relative tosaid at least one surface further comprises a member of the groupconsisting of moving the fluid droplet by altering said magnetic orelectromagnetic field, moving said at least one surface, and acombination thereof.
 55. The method of claim 54, wherein altering amagnetic field comprises altering the position of at least one magnet.56. The method of claim 50, wherein the wettability of said at least onesurface for the fluid of said inner phase of the fluid droplet ischaracterized by an advancing contact angle θ at the interface of afluid droplet, which is made up of the fluid of the inner phase of saidfluid droplet, with said at least one surface of about 50 degrees orhigher.
 57. The method of claim 50, wherein said at least one surface isselected from the group consisting of an essentially flat substrate, aconcave substrate, a convex substrate, and any combination thereof. 58.The method of claim 50, wherein the inner phase of the fluid dropletdirectly contacts matter that is comprised in said surface.
 59. Themethod of claim 50, wherein the fluid of said inner phase of the fluiddroplet is a polar liquid, and said at least one surface is a non-polarsurface.
 60. The method of claim 59, wherein the non-polar surface isselected from the group consisting of silicone, plastic,surface-modified silicon oxide, surface-modified silicon hydride,surface-modified paper, surface-modified glass, surface-modified quartz,surface-modified glimmer, surface-modified metal, surface-modified metaloxide, surface-modified ceramic, and any composite thereof.
 61. Themethod of claim 50, wherein the fluid of said inner phase of the fluiddroplet is a non-polar liquid and said at least one surface is a polarsurface.
 62. The method of claim 50, wherein providing said fluiddroplet comprises: providing a first fluid, providing a second fluidthat is immiscible with the first fluid, and dispensing a droplet of thefirst fluid onto the second fluid, thereby forming a fluid dropletcomprising an inner phase and an outer phase, the first fluid formingthe inner phase, surrounded by the second fluid forming the outer phase.63. The method of claim 62, wherein said first fluid is provided first.64. The method of claim 62, wherein the first fluid, the second fluidand the sample are provided simultaneously.
 65. The method of claim 62,wherein providing said fluid droplet further comprises: collecting saidfluid droplet comprising an inner phase and an outer phase, or a partthereof, out of said second fluid that is forming the outer phase,thereby forming a fluid droplet comprising an outer phase surroundingthe inner phase as a film.
 66. The method of claim 50, wherein the innerphase of the fluid droplet is a polar liquid and the outer phase of thefluid droplet is a non-polar liquid.
 67. The method of claim 66, whereinthe inner phase of the fluid droplet is a hydrophilic liquid and theouter phase of the fluid droplet is a hydrophobic liquid.
 68. The methodof claim 66, wherein the fluid of the inner phase is selected from thegroup consisting of water, deuterium oxide, tritium oxide, an alcohol,an organic acid, an inorganic acid, an ester of an organic acid, anester of an inorganic acid, an ether, an amine, an amide, a nitrile, aketone, an ionic detergent, a non-ionic detergent, carbon dioxide,dimethyl sulfone, dimethyl sulfoxide, a thiol, a disulfide, and a polarionic liquid.
 69. The method of claim 66, wherein the fluid of the outerphase is selected from the group consisting of a mineral oil, a siliconeoil, a natural oil, a perfluorinated carbon liquid, a partiallyhalogenated carbon liquid, an alkane, an alkene, an alkine, an aromaticcompound, carbon disulfide, and a non-polar ionic liquid.
 70. The methodof claim 53, wherein said at least one magnetically attractable particleis selected from the group consisting of a diamagnetic particle, aferromagnetic particle, a paramagnetic particle, and a superparamagneticparticle.
 71. The method of claim 53, wherein said at least onemagnetically attractable particle comprises a ligand that is capable ofbinding target matter suspected to be comprised in said biologicaland/or chemical sample.
 72. The method of claim 71, wherein said ligandis capable of selectively binding target matter suspected to becomprised in said biological and/or chemical sample.
 73. The method ofclaim 71, wherein said ligand is immobilized on the surface of the atleast one magnetically attractable particle.
 74. The method of claim 73,wherein said ligand is selected from the group consisting of a crownether, a peptide, an antibody, a mutein based on a polypeptide of thelipocalin family, a protein based on the ankyrin or crystallinescaffold, an avimer, a glubody, a lectin, a nucleic acid, protein A,protein G, an enzyme, a metal atom, a carbon nanotube, carbon nanofoam,a dye, streptavidin, amylose, maltose, cellulose, chitin, anextracellular matrix, glutathione, calmodulin, gelatine, polymyxin,heparin, NAD, NADP, lysine, arginine, benzamidine and an alumosilicate.75. The method of claim 50, further comprising exposing a region of saidat least one surface to a condition selected from the group consistingof an altered temperature, a magnetic field, an electrical field, anelectromagnetic field, a pressure, a wavelength, a frequency, anamplitude, a chemical concentration and a chemical composition, andwherein controlling the position of said fluid droplet relative to saidat least one surface comprises moving said fluid droplet into the regionof said at least one surface that is being exposed to said condition.76. The method of claim 50, further comprising providing at least twosurfaces that are facing each other, the surfaces being of such atexture and such a wettability for the fluid of said inner phase of thefluid droplet, that the fluid droplet remains intact upon beingcontacted therewith.
 77. The method of claim 76, wherein controlling theposition of said fluid droplet comprises moving said fluid dropletbetween said at least two surfaces by means of said magnetic orelectromagnetic field, and/or by moving at least one of the surfaces.78. The method of claim 50, wherein performing a process on thebiological and/or chemical sample comprises a step selected from thegroup consisting of merging said fluid droplet with a further fluiddroplet, mixing the interior of the fluid droplet, filtering the fluiddroplet through another fluid droplet, and splitting the fluid dropletinto at least two daughter fluid droplets.
 79. The method of claim 50,wherein the biological and/or chemical sample is exposed to a processselected from the group consisting of a physical detection of targetmatter suspected to be comprised in the sample, a chemical reaction, abiochemical reaction, a cell lysis, an extraction of a molecule from anorganism or a part of an organism, and any combination thereof.
 80. Themethod of claim 79, wherein the physical detection reaction is selectedfrom the group consisting of a spectroscopic, a photochemical, aphotometric, a fluorometric, a radiological, an electrical, anacoustical, an electrochemical, a colourimetrical, an interferometrical,a diffractional, and a thermodynamic detection.
 81. The method of claim79, wherein the chemical reaction is selected from the group consistingof a chemical synthesis, a chemical degradation, an enzymatic synthesis,an enzymatic degradation, a chemical modification, an enzymaticmodification, an interaction with a binding molecule, and anycombination thereof.
 82. The method of claim 81, wherein the enzymaticsynthesis is selected from the group consisting of a protein synthesis,a nucleic acid synthesis, a peptide synthesis, a synthesis of apharmaceutical compound, and any combination thereof.
 83. The method ofclaim 50, wherein the sample is selected from the group consisting of asoil sample, an air sample, an environmental sample, a cell culturesample, a bone marrow sample, a rainfall sample, a fallout sample, aspace sample, an extraterrestrial sample, a sewage sample, a groundwater sample, an abrasion sample, an archaeological sample, a foodsample, a blood sample, a serum sample, a plasma sample, a urine sample,a stool sample, a semen sample, a lymphatic fluid sample, acerebrospinal fluid sample, a naspharyngeal wash sample, a sputumsample, a mouth swab sample, a throat swab sample, a nasal swab sample,a bronchoalveolar lavage sample, a bronchial secretion sample, a milksample, an amniotic fluid sample, a biopsy sample, a nail sample, a hairsample, a skin sample, a cancer sample, a tumour sample, a tissuesample, a cell sample, a cell lysate sample, a virus culture sample, aforensic sample, an infection sample, a nosocomial infection sample, aproduction sample, a drug preparation sample, a biological moleculeproduction sample, a protein preparation sample, a lipid preparationsample, a carbohydrate preparation sample, a solution of a nucleotide, asolution of polynucleotide, a solution of a nucleic acid, a solution ofa peptide, a solution of a polypeptide, a solution of an amino acid, asolution of a protein, a solution of a synthetic polymer, a solution ofa biochemical composition, a solution of an organic chemicalcomposition, a solution of an inorganic chemical composition, a solutionof a lipid, a solution of a carbohydrate, a solution of a combinatorychemistry product, a solution of a drug candidate molecule, a solutionof a drug molecule, a solution of a drug metabolite, a suspension of acell, a suspension of a virus, a suspension of a microorganism, asuspension of a metal, a suspension of metal alloy, a solution of ametal ion, and any combination thereof.
 84. A fluid droplet comprisingan inner phase and an outer phase, and at least one magneticallyattractable particle, wherein the outer phase is immiscible with theinner phase, and the outer phase is surrounding the inner phase as afilm and, wherein the inner phase is shielded from the environment bythe outer phase, and wherein said magnetically attractable particlecomprises a ligand that is capable of binding target matter suspected tobe comprised in a biological and/or chemical sample.
 85. The fluiddroplet of claim 84, wherein the fluid of the inner phase of said fluiddroplet is a liquid.
 86. The fluid droplet of claim 84, wherein thefluid of the inner phase of said fluid droplet is a liquid and the fluidof the outer phase of said fluid droplet is a liquid.
 87. The fluiddroplet of claim 84, wherein the inner phase of the fluid droplet is apolar liquid and the outer phase of the fluid droplet is a non-polarliquid.
 88. The fluid droplet of claim 87, wherein the inner phase ofthe fluid droplet is a hydrophilic liquid and the outer phase of thefluid droplet is a hydrophobic liquid.
 89. The fluid droplet of claim87, wherein the fluid of the inner phase is selected from the groupconsisting of water, deuterium oxide, tritium oxide, an alcohol, anorganic acid, an inorganic acid, an ester of an organic acid, an esterof an inorganic acid, an ether, an amine, an amide, a nitrile, a ketone,an ionic detergent, a non-ionic detergent, carbon dioxide, dimethylsulfone, dimethyl sulfoxide, a thiol, a disulfide, and a polar ionicliquid.
 90. The fluid droplet of claim 87, wherein the fluid of theouter phase is selected from the group consisting of a mineral oil, asilicone oil, a natural oil, a perfluorinated carbon liquid, a partiallyhalogenated carbon liquid, an alkane, an alkene, an alkine, an aromaticcompound, carbon disulfide, and a non-polar ionic liquid.
 91. The fluiddroplet of claim 84, wherein said at least one magnetically attractableparticle is selected from the group consisting of a diamagneticparticle, a ferromagnetic particle, a paramagnetic particle, and asuperparamagnetic particle.
 92. The fluid droplet of claim 84, whereinsaid ligand is immobilized on the surface of the at least onemagnetically attractable particle.
 93. The fluid droplet of claim 92,wherein said ligand is selected from the group consisting of a crownether, a peptide, an antibody, a mutein based on a polypeptide of thelipocalin family, a protein based on the ankyrin or crystallinescaffold, an avimer, a glubody, a lectin, a nucleic acid, protein A,protein G, an enzyme, a metal atom, a carbon nanotube, carbon nanofoam,a dye, streptavidin, amylose, maltose, cellulose, chitin, anextracellular matrix, glutathione, calmodulin, gelatine, polymyxin,heparin, NAD, NADP, lysine, arginine, benzamidine and an alumosilicate.94. A method of forming a fluid droplet, which comprises an inner phase,an outer phase and at least one magnetically attractable particle, saidmethod comprising providing a first fluid, providing a second fluid thatis immiscible with the first fluid, contacting the first fluid and thesecond fluid, thereby forming a fluid droplet comprising an inner phaseand an outer phase, the first fluid forming the inner phase, surroundedby the second fluid forming the outer phase, providing at least onemagnetically attractable particle, disposing said at least onemagnetically attractable particle into the fluid droplet, wherein saidmagnetically attractable particle comprises a ligand that is capable ofbinding target matter suspected to be comprised in a biological and/orchemical sample.
 95. The method of claim 94, wherein said first fluid isprovided first.
 96. The method of claim 94, wherein the first fluid andthe second fluid are provided simultaneously.
 97. The method of claim94, wherein the at least one magnetically attractable particle isdisposed into the first fluid before said droplet of the first fluid isdispensed into the second fluid.
 98. The method of claim 94, whereincontacting the first fluid and the second fluid includes dispensing adroplet of the first fluid onto the second fluid.